WO2024059776A2

WO2024059776A2 - Enhanced differentiation of pancreatic islet cells

Info

Publication number: WO2024059776A2
Application number: PCT/US2023/074279
Authority: WO
Inventors: Chunhui XIE; George Harb; Elizabeth RYU; Nicholas TECENO; Christopher FARRAR; Bryce W. CAREY
Original assignee: Vertex Pharmaceuticals Inc
Current assignee: Vertex Pharmaceuticals Inc
Priority date: 2022-09-16
Filing date: 2023-09-15
Publication date: 2024-03-21
Anticipated expiration: 2025-03-16
Also published as: US20250243464A1; WO2024059776A3; EP4587557A2

Abstract

Disclosed herein are compositions and methods related to differentiation of stem cells into pancreatic islet cells. In some aspects, the methods provided herein relate to generation of pancreatic β cell, α cell, δ cells, and EC cells in vitro. In some aspects, the disclosure provides pharmaceutical compositions including the cells generated according to the methods disclosed herein, as well as methods of treatment making use thereof.

Description

ENHANCED DIFFERENTIATION OF PANCREATIC ISLET CELLS

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Patent Application No. 63/376,035, filed September 16, 2022, the contents of which is incorporated herein by reference in its entirely.

BACKGROUND

Transplantation of pancreas or pancreatic islets has been used for treating diabetes, such as type I diabetes. Pancreatic islet transplantation does not need major surgery and the function of the islet grafts can be maintained for years in a recipient. However, a shortage of pancreatic islets donors prevents this therapy from being effectively implemented. Artificial pancreas or pancreatic islets provide an alternative source of transplantable islets. Thus, there is a need for methods of in vitro restitution of pancreatic islets whose function and characteristics resemble endogenous pancreatic islets.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (41583WO_SequenceListing.xml; Size: 36 KB; and Date of Creation: September 11, 2023) is herein incorporated by reference in its entirety.

SUMMARY

Provided herein, in some aspects, are compositions and methods for producing pancreatic islet cells (e.g., stem cell-derived pancreatic islet cells). In some embodiments, a method described herein comprises contacting the population of pluripotent stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, human pluripotent cells) with a medium supplemented with metabolites such as amino acids (e.g., aspartate, glycine or serine), individually, or in combination. Surprisingly, contacting the population of pluripotent stem cells with medium supplemented with metabolites such as amino acids enhanced production of NKX6.1 -positive, ISLl-positive cells (P cells), even when cellular composition during intermediate differentiation stages appear unaffected, relative to cells differentiated in medium without supplementation with metabolites such as amino acids. In some embodiments, a method described herein produces a population of pancreatic cells in which at least 50% of the cells in the population are NKX6.1 -positive, ISLl- positive cells, and wherein less than 20% of the cells are ISL1 -negative cells.

Some aspects of the present disclosure provide in vitro compositions comprising a population of pluripotent stem cells and a medium comprising (i) aspartate at a concentration of at least 120 pM; (ii) glycine at a concentration of at least 40 pM; and/or (iii) serine at a concentration of at least 320 pM.

In some embodiments, the medium further comprises a TGF-P ligand. In some embodiments, the TGF-P ligand is activin A. In some embodiments, the TGF-P ligand (e.g., Activin A) has a concentration of 1-50, 1-25, 5-50, 5-25, 5-15, 8-12, 10-1000, 10- 500, 10-250, 10-125, 75-1000, 75-500, 75-250, 75-125, or 90-110 ng/ml. In some embodiments, the TGF-P ligand (e.g., Activin A) has a concentration of 90-110 ng/ml. In some embodiments, the TGF-P ligand (e.g., Activin A) has a concentration of 8-12 ng/ml.

In some embodiments, the composition further comprises an inhibitor of PI3K/Akt/mTOR signaling. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling comprises one or more of: GSK-690693, IPI-3063, AZD8055, Omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, Nemiralisib, BYL719, FT113, or Apitolisib, or any analog or derivative thereof. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling is GSK-690693 or an analog or a derivative thereof. In some embodiments, the GSK-690693, or an analog or a derivative thereof has a concentration of 0.01-1 pM, 0.02-0.8 pM, 0.05-0.5 pM, 0.06-0.2 pM, 0.07-0.15 pM, or 0.08-0.12 pM.

In some embodiments, the medium further comprises a Wnt signaling pathway activator.

In some embodiments, the Wnt signaling pathway activator is a glycogen synthase kinase 3 (GSK3) inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the Wnt signaling pathway activator has a concentration of 0.1-50, 0.1-25, 0.1-10, 0.1-5, 0.5-50, 0.5-25, 0.5-10, 0.5-5, 1-50, 1-25, 1-10, 1-5, 2-4, or 2-3 pM. In some embodiments, the Wnt signaling pathway activator has a concentration of 2-4 pM.

In some embodiments, the medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the PVA is at most 85% hydrolyzed. In some embodiments, the PVA is about 80% hydrolyzed. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the medium.

In some embodiments, the aspartate has a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM. In some embodiments, the aspartate has a concentration of 200 pM.

In some embodiments, the glycine has a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280- 320, or 150-350 pM. In some embodiments, the glycine has a concentration of 300 pM.

In some embodiments, the serine has a concentration of 320-5000, 320-4000, 320- 3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the serine has a concentration of 585 pM.

In some embodiments, the aspartate has a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and the glycine has a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280- 320 or 150-350. In some embodiments, the aspartate has a concentration of 200 pM and the glycine has a concentration of 300 pM.

In some embodiments, the aspartate has a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and the serine has a concentration of 320-5000, 320-4000, 320- 3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the aspartate has a concentration of 200 pM and serine has a concentration of 585 pM.

In some embodiments, the glycine has a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280- 320, or 150-350 pM and the serine has a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500- 3000, 500-2000, 500-1000, 500-800, 500-600, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the glycine has a concentration of 300 pM and the serine has a concentration of 585 pM.

In some embodiments, the aspartate has a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM, the glycine has a concentration of 40- 600, 40-500, 40-400, 40- 300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200- 400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM, and the serine has a concentration of 320-5000, 320-4000, 320-3000, 320- 2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the aspartate has a concentration of 200 pM, the glycine has a concentration of 300 pM, and the serine has a concentration of 585 pM.

In some embodiments, the in vitro composition further comprises definitive endoderm cells.

In some embodiments, the pluripotent stem cells are embryonic stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are human pluripotent stem cells. In some embodiments, the pluripotent stem cells are genetically modified. In some embodiments, the pluripotent stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, relative to cells that are not genetically modified. In some embodiments, the pluripotent stem cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified. In some embodiments, the pluripotent stem cells are ABO blood group type O. In some embodiments, the pluripotent stem cells have been genetically modified such that the cell is ABO blood group type O. Some aspects of the present disclosure provide methods comprising culturing a first population of cells in a first medium, wherein: the first population of cells comprises pluripotent stem cells; and the first medium comprises: a): (i) aspartate at a concentration of at least 120 pM; (ii) glycine at a concentration of at least 40 pM; and/or (iii) serine at a concentration of at least 320 pM. In some embodiments, the first medium further comprises b): iv) a Wnt signaling pathway activator, v) a transforming growth factor beta ligand and/or vii) an inhibitor of PI3K/Akt/mTOR signaling.

In some embodiments, the first medium further comprises a transforming growth factor beta (TGF-P) ligand. In some embodiments, the TGF-P ligand of the first medium is activin A. In some embodiments, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 1-50, 1-25, 5-50, 5-25, 5-15, 8-12, 10-1000, 10-500, 10-250, 10-125, 75- 1000, 75-500, 75-250, 75-125, or 90-110 ng/ml. In some embodiments, the TGF-P ligand (e.g., Activin A) has a concentration of 90-110 ng/ml. In some embodiments, the TGF-P ligand (e.g., Activin A) has a concentration of 8-12 ng/ml.

In some embodiments, the first medium further comprises an inhibitor of PI3K/Akt/mTOR signaling. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling comprises one or more of: GSK-690693, IPI-3063, AZD8055, Omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, Nemiralisib, BYL719, FT113, or Apitolisib, or any analog or derivative thereof. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling is GSK-690693 or an analog or a derivative thereof. In some embodiments, the GSK-690693, or an analog or a derivative thereof has a concentration of 0.01-1 pM, 0.02-0.8 pM, 0.05-0.5 pM, 0.06-0.2 pM, 0.07-0.15 pM, or 0.08-0.12 pM.

In some embodiments, the first medium further comprises a Wnt signaling pathway activator. In some embodiments, the Wnt signaling pathway activator is a glycogen synthase kinase 3 (GSK3) inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the Wnt signaling pathway activator has a concentration of 0.1-50, 0.1-25, 0.1-10, 0.1-5, 0.5-50, 0.5-25, 0.5-10, 0.5-5, 1-50, 1-25, 1- 10, 1-5, 2-4, or 2-3 pM. In some embodiments, the Wnt signaling pathway activator has a concentration of 2-4 pM.

In some embodiments, the first medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the PVA is at most 85% hydrolyzed. In some embodiments, the PVA is about 80% hydrolyzed. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the first medium.

In some embodiments, the first medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM. In some embodiments, the first medium comprises aspartate at a concentration of 200 pM.

In some embodiments, the first medium comprises glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400- 600, 400-600, 500-600, 280-320, or 150-350 pM. In some embodiments, the first medium comprises glycine at a concentration of 300 pM.

In some embodiments, the first medium comprises serine at a concentration of 320- 5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-400, 320 - 1425, 550-650, or 570-620 pM. In some embodiments, the first medium comprises serine at a concentration of 585 pM.

In some embodiments, the first medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and glycine at a concentration of 40- 600, 40- 500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100- 200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320 or 150-350 pM. In some embodiments, the first medium comprises aspartate at a concentration of 200 pM and glycine at a concentration of 300 pM.

In some embodiments, the first medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500- 5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-400, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the first medium comprises aspartate at a concentration of 200 pM and serine at a concentration of 585 pM. In some embodiments, the first medium comprises glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400- 600, 400-600, 500-600, 280-320, or 150-350 pM and serine at a concentration of 320- 5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, , 500-400, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the first medium comprises glycine at a concentration of 300 pM and serine at a concentration of 585 pM.

In some embodiments, the first medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM, glycine at a concentration of 40- 600, 40- 500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100- 200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500- 4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-400, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the first medium comprises aspartate at a concentration of 200 pM, glycine at a concentration of 300 pM and serine at a concentration of 585 pM.

In some embodiments, the first population of cells are cultured in the first medium for a period of 18-48 hours, resulting in a second population of cells. In some embodiments, the first population of cells are cultured in the first medium for a period of 24 hours, resulting in a second population of cells.

In some embodiments, the method further comprises culturing the second population of cells with a second medium comprising: (i) aspartate at a concentration of at least 120 pM; (ii) glycine at a concentration of at least 40 pM; and/or serine at a concentration of at least 320 pM, wherein the second medium does not comprise a Wnt signaling pathway activator.

In some embodiments, the second medium further comprises a TGF-P ligand. In some embodiments, the TGF-P ligand of the second medium is activin A. In some embodiments, the TGF-P ligand (e.g., Activin A) has a concentration of 1-50, 1-25, 5-50, 5-25, 5-15, 8-12, 10-1000, 10-500, 10-250, 10-125, 75-1000, 75-500, 75-250, 75-125, or 90-110 ng/ml. In some embodiments, the TGF-P ligand (e.g., Activin A) has a concentration of 90-110 ng/ml. In some embodiments, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 8-12 ng/ml.

In some embodiments, the second medium further comprises an inhibitor of PI3K/Akt/mTOR signaling. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling comprises one or more of: GSK-690693, IPI-3063, AZD8055, Omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, Nemiralisib, BYL719, FT113, or Apitolisib, or any analog or derivative thereof. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling is GSK-690693 or an analog or a derivative thereof. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling has a concentration of 0.01-1 pM, 0.02-0.8 pM, 0.05-0.5 pM, 0.06-0.2 pM, 0.07-0.15 pM, or 0.08-0.12 pM.

In some embodiments, the second medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the PVA is at most 85% hydrolyzed. In some embodiments, the PVA is about 80% hydrolyzed. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the second medium.

In some embodiments, the second medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM. In some embodiments, the second medium comprises aspartate at a concentration of 200 pM.

In some embodiments, the second medium comprises glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400- 600, 400-600, 500-600, 280-320, or 150-350 pM. In some embodiments, the second medium comprises glycine at a concentration of 300 pM.

In some embodiments, the second medium comprises serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320- 400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-400, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the second medium comprises serine at a concentration of 585 pM.

In some embodiments, the second medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and glycine at a concentration of 40- 600, 40- 500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100- 200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM. In some embodiments, the second medium comprises aspartate at a concentration of 200 pM and glycine at a concentration of 300 pM.

In some embodiments, the second medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500- 5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-400, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the second medium comprises aspartate at a concentration of 200 pM and serine at a concentration of 585 pM.

In some embodiments, the second medium comprises glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400- 600, 400-600, 500-600, 280-320, or 150-350 pM and serine at a concentration of 320- 5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-400, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the second medium comprises glycine at a concentration of 300 pM and serine at a concentration of 585 pM.

In some embodiments, the second medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM, glycine at a concentration of 40- 600, 40- 500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100- 200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500- 4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-400, 320- 1425, 550-650, or 570-620 pM. In some embodiments, the second medium comprises aspartate at a concentration of 200 pM, glycine at a concentration of 300 pM and serine at a concentration of 585 pM.

In some embodiments, the second population of cells are cultured in the second medium for a period of 36-72 hours, resulting in a third population of cells. In some embodiments, the second population of cells are cultured in the second medium for a period of 48 hours, resulting in a third population of cells.

In some embodiments, the third population of cells comprises definitive endoderm cells. In some embodiments, the method further comprises differentiating the third population of cells into pancreatic endocrine cells. In some embodiments, the pancreatic endocrine cells comprise beta cells, alpha cells, and delta cells.

In some embodiments, the pluripotent stem cells are embryonic stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are human pluripotent stem cells. In some embodiments, the pluripotent stem cells are genetically modified. In some embodiments, the pluripotent stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, relative to cells that are not genetically modified. In some embodiments, the pluripotent stem cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified. In some embodiments, the pluripotent stem cells are ABO blood group type O. In some embodiments, the pluripotent stem cells have been genetically modified such that the cell is ABO blood group type O.

Other aspects of the present disclosure provide in vitro compositions comprising a population of in vitro differentiated cells comprising NKX6.1 -positive, ISLl-positive cells; NKX6.1 -negative, ISLl-positive cells, and ISLl-negative cells, wherein at least 50% of the cells in the population are NKX6.1 -positive, ISLl-positive cells, and wherein less than 20% of the cells are ISLl-negative cells.

In some embodiments, 50%-70% of the cells in the population of in vitro differentiated cells are NKX6.1 -positive, ISLl-positive cells. In some embodiments, up to 30% of the cells in the population of in vitro differentiated cells are NKX6.1 -negative, ISLl-positive cells. In some embodiments, up to 20%-30% of the cells in the population of in vitro differentiated cells are NKX6.1 -negative, ISLl-positive cells.

In some embodiments, the composition comprising a medium. In some embodiments, the medium comprises human serum albumin. In some embodiments, the medium comprises glutamine. In some embodiments, the medium comprises any one or more of the following: an inorganic compound, an Alk5 inhibitor, a thyroid hormone receptor beta-specific agonist, a BMP type I receptor inhibitor, a RHO/ROCK pathway inhibitor, a protein kinase inhibitor, or a S-adenosylhomocysteine hydrolase inhibitor. In some embodiments, the medium comprises any one or more of the following: ZnSCU, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, or DZNEP. In some embodiments, wherein the medium comprises any one or more of L-glutamate, L- carnitine, taurine, acetate, beta-hydroxybutarate, biotin or formate. In some embodiments, the medium comprises some sugar. In some embodiments, the sugar is sucrose or glucose. In some embodiments, the medium comprises the sugar at a concentration of between about 0.05% and about 1.5%. In some embodiments, the medium is a CMRL medium or wherein the medium is HYPOTHERMOSOL® FRS Preservation Media.

In some embodiments, the population of cells are in a cell cluster. In some embodiments, the cell cluster is between 125-225 microns, 130-160, 170-225, 140-200, 140-170, 160-220, 170-215, and 170-200 microns in diameter. In some embodiments, the population comprises cells that are NKX6.1 -positive, ISL1 -positive, and MAFB-positive cells that do not express MAFA.

In some embodiments, wherein the population of cells is derived from pluripotent stem cells in vitro. In some embodiments, the pluripotent stem cells are ABO blood group type O. In some embodiments, the pluripotent stem cells are embryonic stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are human pluripotent stem cells. In some embodiments, the pluripotent stem cells are genetically modified. In some embodiments, the stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, relative to cells that are not genetically modified. In some embodiments, the stem cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified. In some embodiments, the pluripotent stem cells have been genetically modified such that the cell is ABO blood group type O. In some embodiments, the pluripotent stem cells are ABO blood group type O.

In some embodiments, the composition is contained in a device for implantation into a subject.

Further provided herein are implantable encapsulation devices including an internal volume comprising the composition of the present disclosure disposed therein.

In some embodiments, the implantable device comprises at least one membrane that at least partially defines the internal volume. In some embodiments, the at least one membrane includes a first membrane and a second membrane, wherein the first membrane and the second membrane are bonded together to form a seal extending at least partially around the internal volume disposed between the first membrane and the second membrane. In some embodiments, the at least one membrane comprises at least one selected from PVDF, PTFE, ePTFE, PCL, PE/PES, PP, PS, PMMA, PLGA, and PLLA. In some embodiments, the at least one membrane comprises ePTFE. In some embodiments, the device has been implanted in a subject having diabetes. In some embodiments, the subject has Type I Diabetes.

Further provided herein are methods of treating a subject, the method comprising administering to the subject a composition comprising the in vitro composition described herein or implanting the implantable encapsulation device described herein in the subject.

Further provided herein are methods of treating a subject, the method comprising administering to the subject a composition comprising a population in vitro differentiated cells comprising NKX6.1 -positive, ISLl-positive cells; NKX6.1 -negative, ISLl-positive cells, and ISL1 -negative cells, wherein at least 50% of the cells in the population are NKX6.1 -positive, ISLl-positive cells, and wherein less than 20% of the cells are ISL- negative cells.

Further provided herein are methods of treating a subject, the method comprising implanting into the subject an implantable encapsulation device comprising a population in vitro differentiated cells comprising NKX6.1 -positive, ISLl-positive cells; NKX6.1- negative, ISLl-positive cells, and ISL1 -negative cells, wherein at least 50% of the cells in the population are NKX6.1 -positive, ISLl-positive cells, and wherein less than 20% of the cells are ISL-negative cells.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings: FIGs. 1A-1B show graphs of the concentration of glycine at different differentiation stages (In vessel passaging (IVP) cells at a suspension culture passaging stage, Stage 0 cells (StOC), Stage 1 cells (StlC), Stage 2 cells (St2C), Stage 3 cells (St3C), and Stage 4 cells (St4C)). Cells were seeded and differentiated under Protocol 1 and differentiation media was obtained from cells 24-hours after the previous day’s media change. FIG. 1 A shows a graph of the concentration (pg/mL) of glycine observed in 1 mL of fresh media, or in cell differentiation media obtained from cells cultured/differentiated in spinner or bioreactor. FIG. IB shows a graph of metabolite/cell (pg/mM cell) of glycine in cell differentiation media obtained from cells cultured/differentiated in either spinner or bioreactor. Values were obtained by subtracting the concentration of glycine in each sample from the fresh media value for its respective stage. The obtained values were then divided by the viable cell density for each of the samples to generate glycine produced/Mcell (positive values) or consumed/Mcell (negative values) for each sample.

FIGs. 2A-2B shows a graph of fold change values of glycine (FIG. 2A) and non- essential amino acids (e.g., arginine, asparagine, aspartic acid, glycine, proline, serine, tyrosine) (FIG. 2B) observed in cell differentiation media obtained from cells cultured/differentiated in spinner “Spin” or bioreactor “BR” at different differentiation stages, StOC, StlC, St2C, and St3C. The “fresh” media value indicates a control, the dashed line represents the value of fresh media. Any value above the dashed line represents produced glycine/non-essential amino acid and anything below the dashed line represents consumed glycine/non-essential amino acid.

FIGs. 3A-3B shows graphs of the concentration of histidine at different differentiation stages (IVP, StOC, StlC, St2C, St3C, and St4C). Cells were seeded and differentiated under Protocol 1 and differentiation media was obtained from cells 24-hours after the previous day’s media change. FIG. 3 A shows a graph of the concentration (pg/mL) of histidine observed in 1 mL fresh media, or in cell differentiation media obtained from cells cultured/differentiated in spinner or bioreactor. FIG. 3B shows a graph of metabolite/cell (pg/mM cell) of histidine observed in cell differentiation media obtained from cells cultured/differentiated in either spinner “Spin” or bioreactor “BR” at different differentiation stages. Values were obtained by subtracting the concentration of histidine in each sample from the fresh media value for its respective stage. The obtained values were then divided by the viable cell density for each of the samples to generate glycine produced/Mcell (positive values) or consumed/Mcell (negative values) for each sample. FIG. 4 shows a graph of fold change values for essential amino acids (e.g., histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) observed in cell differentiation media obtained from cells cultured/differentiated in spinner “Spin” or bioreactor “BR” at different differentiation stages, StOC, StlC, St2C, and St3C.

FIGs. 5A-5D shows graphs of the concentration of serine and aspartic acid, or graph of metabolite/cell (pg/mM cell) of serine and glycine from cells cultured/differentiated in spinner “Spin” or bioreactor “BR”. The concentration of serine (FIG. 5 A) and aspartic acid (FIG. 5B) was measured at different differentiation stages (IVP, StOC, StlC, St2C, St3C, and St4C) in 1 mL of fresh media, or in cell differentiation media obtained from cells cultured/differentiated in spinner or bioreactor. The observed values of consumed serine (FIG. 5C) and glycine (FIG. 5D) in cell differentiation media at StlC in spinner and a bioreactor.

FIGs. 6A-6E shows results from flow cytometry and quantifications of cells differentiated using either only Protocol 2 (control, “CTL”) or Protocol 2 with supplementation of the indicated amino acids (aspartic acid “Asp”, or glycine “Gly”, or serine “Ser”, or triple combination of aspartic acid, serine and glycine, “AGS”) during SI. Dot plots from flow cytometry analysis of cells that were treated with either control or aspartate supplementation in SI and stained in SIC for Sox 17 and Oct3/4 expression (FIG. 6A) or in S3C for NKX6.1 and PDX1 expression (FIG. 6B) or in S4C for NKX6.1 and PDX1 expression (FIG. 6C) or in S5C for NKX6.1 and ISL1 expression (FIG. 6D). FIG. 6E shows a graph of the percentage of ISL1+/NKX6.1+ cells and ISL1+/NKX6.1- cells (a-like cells) at S5C.

FIG. 7 shows a graph of viable cell density for cell populations treated with either Protocol 2 alone or in combination with one or more amino acid supplemented during SI at each stage of Protocol 2 differentiation (StOC, StlC, St2C, St3C, and St4C).

FIG. 8 shows results from flow cytometry analysis of cells that were treated with either Protocol 2 alone (control) or Protocol 2 supplemented with different combinations of amino acids during SIC and stained for NKX6.1 and ISL1 expression in S5C.

FIGs. 9A-9C show graphs of cell density and the percentage of cells showing a certain genetic marker. FIG. 9A a graph of viable cell density for cell populations treated with Protocol 2 alone (control or “CTL”) or in combination with one or more each amino acid (aspartic acid or “Asp”, glycine or “Gly”, and serine or “Ser”) supplemented during SI at each stage of Protocol 2 differentiation (IVP, StOC, StlC, St2C, St3C, St4C, Stage 5 cells (St5C) and harvest). FIG. 9B shows a graph of the percentage of ISL1+/NKX6.1+ cells and ISL1+/NKX6.1- (a-like) cells at S5C. FIG. 9C shows results from a flow cytometry analysis of cells that were stained for NKX6.1 and ISL1 expression at stage 6, day 11 (“S6D11”).

FIG. 10 shows a graph of viable cell density for cell populations treated with either Protocol 2 alone (control or “CTL”) or in combination with one or more amino acids (aspartic acid or “ASP”, glycine or “GLY”, and serine or “SER”) supplemented during SI at each day of stage 6 in a ds7 differentiation.

FIG. 11 shows a graph of the percentage of ISL1+/NKX6.1+ cells and ISL1+/NKX6.1- (a-like) cells at stage 6, day 11 (“S6D11”) for cell populations treated with either Protocol 2 alone (control or “CTL”) or in combination with one or more amino acids (aspartic acid or “Asp”, glycine or “Gly”, and serine or “Ser”) supplemented during SI.

FIGs. 12A-12B show graphs of P-cell gain in cell populations that were treated with Protocol 2 and supplemented with different combinations of amino acids during stage 1 and quantified at different days (day 4 “D4”, day 7 “D7”, and day 11 “Dl l”) of stage 6 (S6). FIG. 12 A shows a graph of the percentage of net P-cell increase in cell populations treated with Protocol 2 and aspartate/serine supplementation or Protocol 2 and serine/glycine supplementation at different days of the S6 day 4 “S6D4”, S6 day 7 “S6D7”, and S6 day 11 “S6D11”. FIG. 12B shows a graph of the percentage of net P-cell increase (calculated by ISL1+/NKX6.1+ cell% from the control, subtracted from the ISL1+/NKX6.1+ cell% in Ser/Gly SI supplemented cells) at D4, D7 and DI 1 of the S6 differentiation. N=3 differentiations.

FIG. 13 shows a graph of viable cell density at harvest of Protocol 2 differentiation for cell populations treated with Protocol 2 alone (control or “CTL”) or in combination with either aspartate/serine (“ASP/SER”) or serine/glycine (“SER/GLY”) amino acid supplementations during SI.

FIGs. 14A-14C show results from single-cell RNAseq of cells that were treated with Protocol 1. FIG. 14A shows a dot plot of single-cell RNAseq map of cells at SIC of a Protocol 1 differentiation. Each dot is representative of a single cell and Soxl7 gene expression levels are plotted by color intensity. FIG. 14B shows a dot plot of single-cell RNAseq map of cells at SIC of a Protocol 1 differentiation. Each dot is representative of a single cell and is colored to mark cell identity. Cell identity was determined by the expression of HHEX, ID4, P0U5F1, MSX2, SHIS A3, PRTG, KDR and GYPB. FIG. 14C shows a dot plot of gene expression based on cellular identity (e.g., Definitive Endoderm, Early Endoderm, Mesoderm). Dot size indicates the percentage of cells that express each marker while color intensity indicates average gene expression levels.

FIGs. 15A-15D show flow cytometry in dot plots and quantified for cell populations supplemented with amino acids either alone or in combination during Stage 1. FIG. 15A shows flow cytometry results of control and +aspartate/glycine/serine (+AGS) supplemented samples at different days of the Stage 6 differentiation. FIGs. 15B-15D shows graphs of ISL1+/NKX6.1+ (P-like) cells and ISL1+/NKX6.1- (a-like) cells at different days (day 4 “D4” (FIG. 15B), day 7 “D7” (FIG. 15C) and day 11 “Dl l” (FIG. 15D)) of the Stage 6 for each amino acid supplementation.

FIGs. 16A and 16B show graphs of P-cell percentage changes in cell populations that were treated with Protocol 2 and supplemented with aspartate/ serine or serine/glycine during stage 1 and quantified at stage 5 (S5C) or different days (day 4 “D4”, day 7 “D7”, and day 11 “Dl l”) of stage 6 (S6). FIG. 16A shows a graph of the percentage of net P- cell change in cell populations in a spinner. FIG. 16B shows a graph of the percentage of the net P-cell change in cell populations in a bioreactor.

DETAILED DESCRIPTION

The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope.

All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

In this application, the use of “or” means “and/or” unless stated otherwise. The terms “and/or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

The term “diabetes” and its grammatical equivalents as used herein can refer to is a disease characterized by high blood sugar levels over a prolonged period. For example, the term “diabetes” and its grammatical equivalents as used herein can refer to all or any type of diabetes, including, but not limited to, type 1, type 2, cystic fibrosis-related, surgical, gestational diabetes, and mitochondrial diabetes. In some embodiments, diabetes can be a form of hereditary diabetes. In some embodiments, diabetes can be an autoimmune form of diabetes.

The term “endocrine cell(s),” if not particularly specified, can refer to hormone- producing cells present in the pancreas of an organism, such as “islet”, “islet cells”, “islet equivalent”, “islet-like cells”, “pancreatic islets” and their grammatical equivalents. In an embodiment, the endocrine cells can be differentiated from pancreatic progenitor cells or precursors. Islet cells can comprise different types of cells, including, but not limited to, pancreatic a cells, pancreatic P cells, pancreatic 5 cells, pancreatic F cells, and/or pancreatic a cells. Islet cells can also refer to a group of cells, cell clusters, or the like.

The terms “progenitor” and “precursor” cell are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. Often, progenitor cells can also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

A “precursor thereof’ as the term related to an insulin-positive endocrine cell can refer to any cell that is capable of differentiating into an insulin-positive endocrine cell, including for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell, or endocrine progenitor cell, that if cultured under suitable conditions will differentiate the precursor cell into the insulin-positive endocrine cell.

The terms “stem cell-derived P cell,” “SC-P cell,” “functional P cell,” “functional pancreatic P cell,” “mature SC-P cell,” “P-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic P cells) that display at least one marker indicative of a pancreatic P cell (e.g., PDX-1 or NKX6.1), expresses insulin, and display a glucose stimulated insulin secretion (GSIS) response similar or superior to that of an endogenous mature P cell (e.g., a mature P from a healthy functioning pancreas from a healthy adult non-diabetic patient). For simplicity, SC-P cells may be referred to as simply “P cells” in this disclosure. In some embodiments, the terms “SC-P cell” and “non-native P cell” as used herein are interchangeable. In some embodiments, the “SC-P cell” expresses lower levels of MAFA than a pancreatic P cell from a healthy adult human patient. In some embodiments, the “SC-P cell” expresses higher levels of MAFB than a pancreatic P cell from a healthy adult human patient. In some embodiments, the “SC-P cell” expresses higher levels of SIX2, HOPX, IAPP and/or UCN3 than a pancreatic P cell from a healthy adult human patient. In some embodiments, the “SC-P cell” comprises a mature pancreatic cell. It is to be understood that the SC-P cells need not be derived (e.g., directly) from stem cells, as the methods of the disclosure are capable of deriving SC-P cells from any insulin-positive endocrine cell or precursor thereof using any cell as a starting point (e.g., one can use embryonic stem cells, induced-pluripotent stem cells, progenitor cells such as definitive endoderm cells, partially reprogrammed somatic cells (e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells, etc., as the invention is not intended to be limited in this manner). In some embodiments, the SC-P cells exhibit a response to multiple glucose challenges (e.g., at least one, at least two, or at least three or more sequential glucose challenges). In some embodiments, the response resembles the response of endogenous islets (e.g., human islets) to multiple glucose challenges. In some embodiments, the morphology of the SC-P cell resembles the morphology of an endogenous P cell. In some embodiments, the SC-P cell exhibits an in vitro GSIS response that resembles the GSIS response of an endogenous P cell. In some embodiments, the SC-P cell exhibits an in vivo GSIS response that resembles the GSIS response of an endogenous P cell. In some embodiments, the SC-P cell exhibits both an in vitro and in vivo GSIS response that resembles the GSIS response of an endogenous P cell. In some embodiments, the GSIS response of the SC-P cell can be observed within two weeks of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-P cell can be observed within three weeks of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-P cell can be observed within four weeks of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-P cell can be observed between one month and three months of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the SC-P cells package insulin into secretory granules. In some embodiments, the SC-P cells exhibit encapsulated crystalline insulin granules. In some embodiments, the SC-P cells exhibit a stimulation index of greater than 1. In some embodiments, the SC-P cells exhibit a stimulation index of greater than 1.1. In some embodiments, the SC-P cells exhibit a stimulation index of greater than 2. In some embodiments, the stimulation index of the cell is characterized by the ratio of insulin secreted in response to high glucose concentrations (e.g., 15 mM) compared to low glucose concentrations (e.g., 2.5 mM).

In some embodiments, the SC-P cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from the SC-P cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-P cells are monohormonal. In some embodiments, the SC-P cells do not abnormally co-express other hormones, such as glucagon, somatostatin or pancreatic polypeptide. In some embodiments, the SC-P cells exhibit a low rate of replication. In some embodiments, the SC-P cells increase intracellular Ca2+ in response to glucose.

The terms “stem cell-derived a cell,” “SC-a cell,” “functional a cell,” “functional pancreatic a cell,” “mature SC-a cell,” “a-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic a cells) that display at least one marker indicative of a pancreatic a cell (e.g., glucagon, expressing ISL1 but not NKX6.1), expresses glucagon, and is capable of secreting functional glucagon in response to a stimulus that induces an endogenous pancreatic a cell to secrete functional glucagon. In some embodiments, the “SC-a cell” does not express somatostatin. In some embodiments, the “SC-a cell” does not express insulin. In some embodiments, the terms “SC-a cell” and “non-native a cell” as used herein are interchangeable. In some embodiments, the “SC-a cell” comprises a mature pancreatic cell. For short, these cells may be referred to as simply “a cells” in this disclosure.

The terms “stem cell-derived 5 cell,” “SC-5 cell,” “functional 5 cell,” “functional pancreatic 5 cell,” “mature SC-5 cell,” “5-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic 5 cells) that display at least one marker indicative of a pancreatic 5 cell (e.g., somatostatin), expresses and is capable of secreting somatostatin in response to a stimulus that induces an endogenous pancreatic 5 cell to secrete functional glucagon. For simplicity, SC- 5 cells may be referred to as simply “5 cells” in this disclosure. In some embodiments, “SC-5 cell” does not express glucagon. In some embodiments, “SC-5 cell” does not express insulin. In some embodiments, the terms “SC-5 cell” and “non-native 5 cell” as used herein are interchangeable. In some embodiments, the “SC-5 cell” comprises a mature pancreatic cell.

The terms “stem cell-derived enterochromaffin (EC) cell,” “SC-EC cell,” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic EC cells) that display at least one marker indicative of a pancreatic EC cell (e.g., VMAT1 (vesicular monoamine transporter 1), expressing NKX6.1 but not ISL1). In some embodiments, the terms “SC-EC cell” and “non-native EC cell” as used herein are interchangeable.

Similar to SC-P cells, it is to be understood that the SC-a, SC-5 cells, and SC-EC cells need not be derived (e.g., directly) from stem cells, as the methods of the disclosure are capable of deriving SC-a cells from other precursor cells generated during in vitro differentiation of SC-P cells as a starting point (e.g., one can use embryonic stem cells, induced-pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells, etc., as the invention is not intended to be limited in this manner).

As used herein, the term “insulin producing cell” and its grammatical equivalent refer to a cell differentiated from a pancreatic progenitor, or precursor thereof, which secretes insulin. An insulin-producing cell can include pancreatic P cell as that term is described herein, as well as pancreatic P-like cells (e.g., insulin-positive, endocrine cells) that synthesize (e.g., transcribe the insulin gene, translate the proinsulin mRNA, and modify the proinsulin mRNA into the insulin protein), express (e.g., manifest the phenotypic trait carried by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. A population of insulin producing cells e.g., produced by differentiating insulin-positive endocrine cells or a precursor thereof into SC-P cells according to the methods of the present disclosure can be pancreatic P cells or P-like cells (e.g., cells that have at least one, or at least two least characteristics of an endogenous P cell and exhibit a glucose stimulated insulin secretion (GSIS) response that resembles an endogenous adult P cell). The population of insulinproducing cells, e.g., produced by the methods as disclosed herein can comprise mature pancreatic P cell or SC-P cells, and can also contain non-insulin-producing cells (e.g., cells of cell like phenotype with the exception they do not produce or secrete insulin).

The terms “insulin-positive P-like cell,” “insulin-positive endocrine cell,” and their grammatical equivalents can refer to cells (e.g., pancreatic endocrine cells) that display at least one marker indicative of a pancreatic P cell and also expresses insulin but, unless specified otherwise, lack a glucose stimulated insulin secretion (GSIS) response characteristic of an endogenous P cell. Exemplary markers of “insulin-positive endocrine cell” include, but are not limited to, NKX6.1 (NK6 homeobox 1), ISL1 (Isletl), and insulin.

The term “P cell marker” refers to, without limitation, proteins, peptides, nucleic acids, polymorphism of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analyte which are expressed or present in pancreatic P cells. Exemplary p cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (PDX1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3p, PCV3, B2, Nkx2.2, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Inf lb, Hnf-6, Hnf-3beta, VMAT2, NKX6.1, and MafA, and those described in Zhang et al., Diabetes. 50(10):2231- 6 (2001). In some embodiments, the P cell marker is a nuclear P-cell marker. In some embodiments, the P cell marker is PDX1 or PH3.

The term “pancreatic endocrine marker” can refer to without limitation, proteins, peptides, nucleic acids, polymorphism of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes which are expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD and Islet-1.

The term “pancreatic progenitor,” “pancreatic endocrine progenitor,” “pancreatic precursor,” “pancreatic endocrine precursor” and their grammatical equivalents are used interchangeably herein and can refer to a stem cell which is capable of becoming a pancreatic hormone expressing cell capable of forming pancreatic endocrine cells, pancreatic exocrine cells or pancreatic duct cells. These cells are committed to differentiating towards at least one type of pancreatic cell, e.g. p cells that produce insulin; a cells that produce glucagon; 5 cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide. Such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

The term “PDX1 -positive pancreatic progenitor” as used herein can refer to a cell which is a pancreatic endoderm (PE) cell which has the capacity to differentiate into SC-P cells, such as pancreatic P cells. A PDX1 -positive pancreatic progenitor expresses the marker PDX1. Other markers include, but are not limited to Cdcpl, or Ptfl a, or HNF6 or NRx2.2. The expression of PDX1 may be assessed by any method known by the skilled person such as immunochemistry using an anti-PDXl antibody or quantitative RT-PCR. In some embodiments, a PDX1 -positive pancreatic progenitor cell lacks expression of NKX6.1. In some embodiments, a PDXl-positive pancreatic progenitor cell can also be referred to as PDXl-positive, NKX6.1 -negative pancreatic progenitor cell due to its lack of expression of NKX6.1. In some embodiments, the PDXl-positive pancreatic progenitor cells can also be termed as “pancreatic foregut endoderm cells.”

The terms “PDXl-positive, NKX6.1 -positive pancreatic progenitor,” and “NKX6.1 -positive pancreatic progenitor” are used interchangeably herein and can refer to a cell which is a pancreatic endoderm (PE) cell which has the capacity to differentiate into insulin-producing cells, such as pancreatic P cells. A PDXl-positive, NKX6.1 -positive pancreatic progenitor expresses the markers PDX1 and NKX6-1. Other markers may include, but are not limited to Cdcpl, or Ptfl a, or HNF6 or NRx2.2. The expression of NKX6-1 may be assessed by any method known by the skilled person such as immunochemistry using an anti-NKX6-l antibody or quantitative RT-PCR. As used herein, the terms “NKX6.1” and “NKX6-1” are equivalent and interchangeable. In some embodiments, the PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells can also be termed as “pancreatic foregut precursor cells.”

The terms “NeuroD” and “NeuroDl” are used interchangeably and identify a protein expressed in pancreatic endocrine progenitor cells and the gene encoding it.

The term “differentiated cell” or its grammatical equivalents means any primary cell that is not, in its native form, pluripotent as that term is defined herein. Stated another way, the term “differentiated cell” can refer to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell such as an induced pluripotent stem cell) in a cellular differentiation process. Without wishing to be limited to theory, a pluripotent stem cell in the course of normal ontogeny can differentiate first to an endoderm cell that is capable of forming pancreas cells and other endoderm cell types. Further differentiation of an endoderm cell may lead to the pancreatic pathway, where -98% of the cells become exocrine, ductular, or matrix cells, and -2% become endocrine cells. Early endocrine cells are islet progenitors, which can then differentiate further into insulin-producing cells (e.g. functional endocrine cells) which secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endoderm cells can also be differentiated into other cells of endodermal origin, e.g. lung, liver, intestine, thymus etc.

As used herein, the term “somatic cell” can refer to any cells forming the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as “gametes”) are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body - apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells - is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. In some embodiments the somatic cell is a “non-embryonic somatic cell”, by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell”, by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro. Unless otherwise indicated the methods for converting at least one insulin-positive endocrine cell or precursor thereof to an insulin-producing, glucose responsive cell can be performed both in vivo and in vitro (where in vivo is practiced when at least one insulin-positive endocrine cell or precursor thereof are present within a subject, and where in vitro is practiced using an isolated at least one insulin-positive endocrine cell or precursor thereof maintained in culture).

As used herein, the term “adult cell” can refer to a cell found throughout the body after embryonic development.

The term “endoderm cell” as used herein can refer to a cell which is from one of the three primary germ cell layers in the very early embryo (the other two germ cell layers are the mesoderm and ectoderm). The endoderm is the innermost of the three layers. An endoderm cell differentiates to give rise first to the embryonic gut and then to the linings of the respiratory and digestive tracts (e.g., the intestine), the liver and the pancreas.

The term “a cell of endoderm origin” as used herein can refer to any cell which has developed or differentiated from an endoderm cell. For example, a cell of endoderm origin includes cells of the liver, lung, pancreas, thymus, intestine, stomach and thyroid. Without wishing to be bound by theory, liver and pancreas progenitors (also referred to as pancreatic progenitors) are developed from endoderm cells in the embryonic foregut. Shortly after their specification, liver and pancreas progenitors rapidly acquire markedly different cellular functions and regenerative capacities. These changes are elicited by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in the development and regeneration of the organs has been fueled by the intense need for hepatocytes and pancreatic P cells in the therapeutic treatment of liver failure and type I diabetes. Studies in diverse model organisms and humans have revealed evolutionarily conserved inductive signals and transcription factor networks that elicit the differentiation of liver and pancreatic cells and provide guidance for how to promote hepatocyte and P cell differentiation from diverse stem and progenitor cell types.

The term “definitive endoderm” as used herein can refer to a cell differentiated from an endoderm cell and which can be differentiated into a SC-P cell (e.g., a pancreatic P cell). A definitive endoderm cell expresses the marker Soxl7. Other markers characteristic of definitive endoderm cells may include, but are not limited to MIXL2, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, 0TX2, goosecoid, C-Kit, CD99, CMK0R1 and CRIP1. In particular, definitive endoderm cells herein express Sox 17 and in some embodiments Sox 17 and HNF3B, and do not express significant levels of GATA4, SPARC, APF or DAB. Definitive endoderm cells are not positive for the marker PDX1 e.g. they are PDX1 -negative). Definitive endoderm cells have the capacity to differentiate into cells including those of the liver, lung, pancreas, thymus, intestine, stomach and thyroid. The expression of Sox 17 and other markers of definitive endoderm may be assessed by any method known by the skilled person such as immunochemistry, e.g., using an anti-Soxl7 antibody, or quantitative RT-PCR.

The term “pancreatic endoderm” can refer to a cell of endoderm origin which is capable of differentiating into multiple pancreatic lineages, including pancreatic P cells, but no longer has the capacity to differentiate into non-pancreatic lineages.

The term “pancreatic islet cells” refers to a population of cells that include different types of pancreatic endocrine cells (P-cells, a-cells, 5-cells, s-cells) and enterochromaffin (EC) cells, e.g., as described in Xavier et al. (J Clin Med. 2018 Mar; 7(3): 54), incorporated herein by reference.

The term “primitive gut tube cell” or “gut tube cell” as used herein can refer to a cell differentiated from an endoderm cell and which can be differentiated into a SC-P cell {e.g., a pancreatic P cell). A primitive gut tube cell expresses at least one of the following markers: HNP1-P, HNF3-P or HNF4-a. In some embodiments, a primitive gut tube cell is FOXA2-positive and SOX2 -positive, i.e., expresses both FOXA2 (also known as HNF3-P) and SOX2. In some embodiments, a primitive gut tube cell is FOXA2 -positive and PDX1 -negative, i.e., expresses FOXA2 but not PDX1. Primitive gut tube cells have the capacity to differentiate into cells including those of the lung, liver, pancreas, stomach, and intestine. The expression of HNF1-P and other markers of primitive gut tube may be assessed by any method known by the skilled person such as immunochemistry, e.g., using an anti-HNFl-P antibody.

The term “phenotype” can refer to one or a number of total biological characteristics that define the cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. The “non-human animals” and “nonhuman mammals” as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g. cow, sheep, pig, and the like. “Patient in need thereof’ or “subject in need thereof’ is referred to herein as a patient diagnosed with or suspected of having a disease or disorder, for instance, but not restricted to diabetes.

“Administering” as used herein can refer to providing one or more compositions described herein to a patient or a subject. By way of example and not limitation, composition administration, e.g., injection, can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by the oral route. Additionally, administration can also be by surgical deposition of a bolus or pellet of cells, or positioning of a medical device. In an embodiment, a composition of the present disclosure can comprise engineered cells or host cells expressing nucleic acid sequences described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount that is effective to treat or prevent proliferative disorders. A pharmaceutical composition can comprise the cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

The ranges disclosed throughout are sometimes referred to as, for example, “X is administered on or on about day 1 to 2; or 2 to 3 [or any numerical range].” This range includes the numbers themselves (e.g., the endpoints of the range) and any individual numbers present in this range.

All these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary. Differentiation stages

Pancreatic differentiation as disclosed herein may be carried out in a step-wise manner. In an exemplary embodiment of the step-wise progression, “Stage 1” or “SI” or “Stl” refers to the first step in the differentiation process, the differentiation of pluripotent stem cells into cells expressing markers characteristic of definitive endoderm cells (“DE”, “Stage 1 cells” or “Stl cells” or “SI cells”). “Stage 2” refers to the second step, the differentiation of cells expressing markers characteristic of definitive endoderm cells into cells expressing markers characteristic of gut tube cells (“GT”, “Stage 2 cells” “St2 cells” or “S2 cells”). “Stage 3” refers to the third step, the differentiation of cells expressing markers characteristic of gut tube cells into cells expressing markers characteristic of pancreatic progenitor 1 cells (“PPI”, “Stage 3 cells” or “ St3 cells” or “S3 cells”). “Stage 4” refers to the fourth step, the differentiation of cells expressing markers characteristic of pancreatic progenitor 1 cells into cells expressing markers characteristic of pancreatic progenitor 2 cells (“PP2”, “Stage 4 cells” or “St4 cells” or “S4 cells”). “Stage 5” refers to the fifth step, the differentiation of cells expressing markers characteristic of pancreatic progenitor 2 cells (e.g., PDX.1+, NKX6.1+) into cells expressing markers characteristic of pancreatic endoderm cells and/or pancreatic endocrine progenitor cells (e.g., insulin+) (“EN”, “Stage 5 cells” or “St5 cells” or “S5 cells”). “Stage 6” refers to the differentiation of cells expressing markers characteristic of pancreatic endocrine progenitor cells (e.g., insulin) into cells expressing markers characteristic of pancreatic endocrine P cells (“SC-P cells”) or pancreatic endocrine a cells (“SC-a cells”). It should be appreciated, however, that not all cells in a particular population progress through these stages at the same rate, i.e., some cells may have progressed less, or more, down the differentiation pathway than the majority of cells present in theparticular population. For example, in some embodiments, SC-P cells can be identified during stage 5, at the conclusion of stage 5, at the beginning of stage 6, etc. Examples of methods of making cells of any one of stages 1- 6 are provided in, for example, US Patent 10,030,229; US Patent 10,443,042; published application US 20200332262; and published application US 20210198632, published application US 20220090020, and published application WO2022147056, each of which is incorporated by reference in its entirety. Compositions and methods for producing pancreatic islet cells

In some aspects, the present disclosure provides compositions and methods of differentiating pancreatic islet cells (e.g., differentiating from stem cells such as human embryonic stem cells or human pluripotent stem cells). The compositions and methods provided herein can, in some embodiments, offer pancreatic SC-islet cells, cell populations, or cell clusters containing pancreatic SC-P cells and pancreatic SC-a cells. In some embodiments, such pancreatic SC-islet cells, cell populations or cell clusters exhibit, high insulin content, superior glucose-dependent insulin secretion response, as well as a percentage of pancreatic SC-a, SC-P, and SC-5 cells and enterochromaffin (EC) cells, which can resemble native pancreatic islets both structurally and functionally. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein comprises at least 50% pancreatic SC-P cells, up to 30% pancreatic SC-a cells, 3-10% pancreatic SC-5 cells, and/or less than SC-20% EC cells. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein has improved glucose-stimulated insulin secretion (GSIS) response as compared to cell compositions generated according to conventional methods. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein has dynamic GSIS response similar to native pancreatic islets (e.g., pancreatic islets from a healthy functioning pancreas from a healthy adult non-diabetic subject).

In some embodiments, a method of producing pancreatic islet cells (e.g., SC-beta cells, SC-alpha cells, SC-delta cells) described herein comprises contacting pluripotent stem cells (e.g., human embryonic stem cells or induced pluripotent stem cells) with a medium supplemented with additional metabolites, such as amino acids (e.g., aspartate, glycine, and/or serine). In some embodiments, a method of producing pancreatic islet cells (e.g., SC-beta cells, SC-alpha cells, SC-delta cells) described herein comprises contacting pluripotent stem cells (e.g., human embryonic stem cells or induced pluripotent stem cells) with a medium supplemented with additional amino acids (e.g., aspartate, glycine, and/or serine) and further comprising a TGF-P ligand (e.g., activin A), a Wnt signaling pathway activator (e.g., CHIR99021), and/or an inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693). In some embodiments, a method of producing pancreatic islet cells (e.g., SC-beta cells, SC-alpha cells, SC-delta cells) described herein comprises contacting pluripotent stem cells (e.g., human embryonic stem cells or induced pluripotent stem cells) with a medium supplemented with additional amino acids (e.g., aspartate, glycine, and/or serine) and further comprising a TGF-P ligand (e.g., activin A) and a Wnt signaling pathway activator (e.g., CHIR99021), and optionally an inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693).

In some embodiments, a method of producing pancreatic islet cells (e.g., SC-beta cells, SC-alpha cells, SC-delta cells) described herein comprises contacting pluripotent stem cells (e.g., human embryonic stem cells or induced pluripotent stem cells) with a medium supplemented with additional amino acids (e.g., aspartate, glycine, and/or serine) and further comprising a TGF-P ligand (e.g., activin A) and/or a Wnt signaling pathway activator (e.g., CHIR99021), and optionally an inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693) for a first period of time, followed by contacting the result cells with a medium supplemented with additional amino acids (e.g., aspartate, glycine, and/or serine) and further comprising a TGF-P ligand (e.g., activin A) and optionally an inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693), but no Wnt signaling pathway activator. In some embodiments, such contacting differentiates the pluripotent stem cells (e.g., human embryonic stem cells or induced pluripotent stem cells) to definitive endoderm cells, which may be further differentiated into pancreatic islet cells (e.g., SC-beta cells, SC-alpha cells, SC-delta cells) using any of the differentiation methods described herein or known in the art.

Composition comprising pluripotent stem cells and amino acids

In some aspects, the present disclosure provides in vitro compositions comprising a population of pluripotent stem cells (e.g., human embryonic stem cells or induced pluripotent stem cells) and a medium supplemented with additional metabolites (e.g., additional amino acids). It is to be understood that a base medium (e.g., a commercially available medium such as MCDB 131 Medium, Signa-Aldrich) contains a base level of metabolites and amino acids. A medium used in a method described herein may be supplemented with additional metabolites (e.g., amino acids), which, in some embodiments, results in a higher concentration of certain metabolites (e.g., amino acids (e.g., aspartate, glycine, and/or serine)) than the base level in the base medium.

In some embodiments, the medium further comprises one or more (e.g., 1, 2, 3, 4 or more) agents selected from: a TGF-P ligand (e.g., activin A), an inhibitor of PI3K/Akt/mT0R signaling (e.g., GSK-690693), a Wnt signaling pathway activator (e.g., CHIR99021), and a water-soluble synthetic polymer (e.g., PVA). In some embodiments, the medium further comprises a TGF-P ligand (e.g., activin A). In some embodiments, the medium further comprises a TGF-P ligand (e.g., activin A) and a Wnt signaling pathway activator (e.g., CHIR99021). In some embodiments, the medium further comprises a TGF-P ligand (e.g., activin A), a Wnt signaling pathway activator (e.g., CHIR99021), and a water-soluble synthetic polymer (e.g., PVA). In some embodiments, the medium further comprises a TGF-P ligand (e.g., activin A), an inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693), and a Wnt signaling pathway activator (e.g., CHIR99021). In some embodiments, the medium further comprises a TGF-P ligand (e.g., activin A), an inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693), a Wnt signaling pathway activator (e.g., CHIR99021), and a water-soluble synthetic polymer (e.g., PVA). In some embodiments, the medium does not comprise a Wnt signaling pathway activator (e.g., CHIR99021).

In some embodiments, the medium of an in vitro composition described herein comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM); (ii) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM); and/or (iii) serine at a concentration of higher than 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, the medium of an in vitro composition described herein comprises aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM). In some embodiments, the medium of an in vitro composition described herein comprises glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM). In some embodiments, the medium of an in vitro composition described herein comprises serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, the medium of an in vitro composition described herein comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM); and (ii) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM). In some embodiments, the medium of an in vitro composition described herein comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM), and (ii) serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM). In some embodiments, the medium of an in vitro composition described herein comprises (i) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM); and (ii) serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, the medium of an in vitro composition described herein comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM); (ii) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM); and (iii) serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, the medium of an in vitro composition described herein comprises aspartate, wherein the aspartate has a concentration of about 100-1000 pM (e.g., 100-1000, 100-800, 100-500, 100-400, 100-300, 100-250, 100-220, 100-210, 100- 200, 100-190, 100-160, 100-120, 120-1000, 120-800, 120-500, 120-400, 120-300, 120- 250, 120-220, 120-210, 120-200, 120-190, 120-160, 160-1000, 160-800, 160-500, 160-

400, 160-300, 160-250, 160-220, 160-210, 160-200, 160-190, 190-1000, 190-800, 190-

500, 190-400, 190-300, 190-250, 190-220, 190-210, 190-200, 200-1000, 200-800, 200-

500, 200-400, 200-300, 200-250, 200-220, 200-210, 210-1000, 210-800, 210-500, 210-

400, 210-300, 210-250, 210-220, 220-1000, 220-800, 220-500, 220-400, 220-300, 220- 250, 250-1000, 250-800, 250-500, 250-400, 250-300, 300-1000, 300-800, 300-500, 300- 400, 400-1000, 400-800, 400-500, 500-1000, 500-800, 800-1000 pM). In some embodiments, the aspartate has a concentration of about 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM. In particular embodiments, the aspartate has a concentraton of about 190- 210 pM. In some embodiments, the aspartate has a concentration of about 100, 120, 160, 190, 200, 210, 220, 250, 300, 400, 500, 800, 1000 pM. In some embodiments, the aspartate has a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10 or more than 100 pM). In some embodiments, the aspartate has a concentration of about 200 pM.

In some embodiments, the medium of an in vitro composition described herein comprises glycine, wherein the glycine has a concentration of about 30-600 pM (e.g., 30- 600, 30-500, 30-400, 30-350, 30-320, 30-300, 30-280, 30-200, 30-150, 30-100, 30-80, 30- 40, 40-600, 40-500, 40-400, 40-350, 40-320, 40-300, 40-280, 40-200, 40-150, 40-100, 40- 80, 80-600, 80-500, 80-400, 80-350, 80-320, 80-300, 80-280, 80-200, 80-150, 80-100, 100-600, 100-500, 100-400, 100-350, 100-320, 100-300, 100-280, 100-200, 100-150, 150- 600, 150-500, 150-400, 150-350, 150-320, 150-300, 150-280, 150-200, 200-600, 200-500, 200-400, 200-350, 200-320, 200-300, 200-280, 280-600, 280-500, 280-400, 280-350, 280- 320, 280-300, 300-600, 300-500, 300-400, 300-350, 300-320, 320-600, 320-500, 320-400, 320-350, 350-600, 350-500, 350-400, 400-600, 400-500, 500-600 pM). In some embodiments, the glycine has a concentration of about 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM. In particular embodiments, the glycine has a concentration of about 280-320 pM. In some embodiments, the glycine has a concentration of about 30, 40, 80, 100, 150, 200, 280, 300, 320, 350, 400, 500, 600 pM. In some embodiments, the glycine has a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10 or more than 30 pM). In some embodiments, the glycine has a concentration of about 300 pM.

In some embodiments, the medium of an in vitro composition described herein comprises serine, wherein the serine has a concentration of about 285-5000 pM (e.g., 285- 5000, 285-4000, 285-3000, 285-2000, 285-1425, 285-1000, 285-800, 285-650, 285-620, 285-600, 285-585, 285-570, 285-550, 285-500, 285-400, 285-320, 320-5000, 320-4000, 320-3000, 320-2000, 320-1425, 320-1000, 320-800, 320-650, 320-620, 320-600, 320-585, 320-570, 320-550, 320-500, 320-400, 400-5000, 400-4000, 400-3000, 400-2000, 400- 1425, 400-1000, 400-800, 400-650, 400-620, 400-600, 400-585, 400-570, 400-550, 400- 500, 500-5000, 500-4000, 500-3000, 500-2000, 500-1425, 500-1000, 500-800, 500-650, 500-620, 500-600, 500-585, 500-570, 500-550, 550-5000, 550-4000, 550-3000, 550-2000, 550-1425, 550-1000, 550-800, 550-650, 550-620, 550-600, 550-585, 550-570, 570-5000, 570-4000, 570-3000, 570-2000, 570-1425, 570-1000, 570-800, 570-650, 570-620, 570- 600, 570-585, 585-5000, 585-4000, 585-3000, 585-2000, 585-1425, 585-1000, 585-800, 585-650, 585-620, 585-600, 600-5000, 600-4000, 600-3000, 600-2000, 600-1425, 600- 1000, 600-800, 600-650, 600-620, 620-5000, 620-4000, 620-3000, 620-2000, 620-1425, 620-1000, 620-800, 620-650, 650-5000, 650-4000, 650-3000, 650-2000, 650-1425, 650- 1000, 650-800, 800-5000, 800-4000, 800-3000, 800-2000, 800-1425, 800-1000, 1000- 5000, 1000-4000, 1000-3000, 1000-2000, 1000-1425, 1425-5000, 1425-4000, 1425-3000, 1425-2000, 2000-5000, 2000-4000, 2000-3000, 3000-5000, 3000-4000, 4000-5000 pM). In some embodiments, the serine has a concentration of about 320-5000, 320-4000, 320- 3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550- 650, or 570-620 pM. In some embodiments, the serine has a concentration of 570-620 pM. In some embodiments, the serine has a concentration of about 285, 320, 400, 500, 550, 570, 585, 600, 620, 650, 800, 1000, 1425, 2000, 3000, 4000, 5000 pM. In some embodiments, the medium comprises serine of a concentration at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10 or more than 285 pM). In some embodiments, the serine has a concentration about 585 pM.

In some embodiments, the medium of an in vitro composition described herein comprises aspartate and glycine, wherein the aspartate has a concentration of about 100- 1000 pM (e.g., 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160- 300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM) and wherein the glycine has a concentration of about 30-600 pM (e.g., 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200- 300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM). In some embodiments, the aspartate has a concentration of about 200 pM and the glycine has a concentration of about 300 pM.

In some embodiments, the medium of an in vitro composition described herein comprises aspartate and serine, wherein the aspartate has a concentration of about 100- 1000 pM (e.g., 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160- 300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM) and the serine has a concentration of about 285-5000 pM (320-5000, 320-4000, 320-3000, 320-2000, 320- 1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550-650, or 570-620 pM). In some embodiments, the aspartate has a concentration of about 200 pM and the serine has a concentration of about 585 pM.

In some embodiments, the medium of an in vitro composition described herein comprises glycine and serine, wherein the glycine has a concentration of about 30-600 pM (e.g., 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400- 600, 400-600, 500-600, 280-320, or 150-350 pM) and the serine has a concentration of about 285-5000 pM (320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320- 600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550-650, or 570-620 pM). In some embodiments, the glycine has a concentration of about 300 pM and the serine has a concentration of about 585 gM.

In some embodiments, the medium of an in vitro composition described herein comprises aspartate, glycine, and serine, wherein the aspartate has a concentration of about 100-1000 pM (e.g., 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM), the glycine has a concentration of about 30-600 pM (e.g., 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200- 300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM), and the serine has a concentration of about 285-5000 pM (320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500- 3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550-650, or 570-620 pM). In one embodiment, the aspartate has concentration of about 200 pM, the glycine has a concentration of about 300 pM, and the serine has a concentration of about 585 pM.

In some embodiments, the medium of an in vitro composition described herein further comprises a TGF-P ligand (e.g., activin A). In some embodiments, the TGF-P ligand (e.g., activin A) has a concentration of about 1-200 ng/ml (e.g., 1-200, 1-150, 1- 125, 1-110, 1-100, 1-90, 1-75, 1-50, 1-25, 1-15, 1-12, 1-10, 1-8, 1-5, 5-200, 5-150, 5-125, 5-110, 5-100, 5-90, 5-75, 5-50, 5-25, 5-15, 5-12, 5-10, 5-8, 8-200, 8-150, 8-125, 8-110, 8- 100, 8-90, 8-75, 8-50, 8-25, 8-15, 8-12, 8-10, 10-200, 10-150, 10-125, 10-110, 10-100, 10- 90, 10-75, 10-50, 10-25, 10-15, 10-12, 12-200, 12-150, 12-125, 12-110, 12-100, 12-90, 12-75, 12-50, 12-25, 12-15, 15-200, 15-150, 15-125, 15-110, 15-100, 15-90, 15-75, 15-50, 15-25, 25-200, 25-150, 25-125, 25-110, 25-100, 25-90, 25-75, 25-50, 50-200, 50-150, SO- 125, 50-110, 50-100, 50-90, 50-75, 75-200, 75-150, 75-125, 75-110, 75-100, 75-90, 90- 200, 90-150, 90-125, 90-110, 90-100, 100-200, 100-150, 100-125, 100-110, 110-200, 110- 250, 110-125, 125-200, 125-150, or 150-200 ng/ml). In some embodiments, the TGF-P ligand (e.g., activin A) has a concentration of about 1-50, 1-25, 5-50, 5-25, 5-15, 8-12, 10- 1000, 10-500, 10-250, 10-125, 75-1000, 75-500, 75-250, 75-125, or 90-110 ng/ml. In some embodiments, the TGF-P ligand (e.g., activin A) has a concentration of about 90-110 ng/ml (e.g., 90, 95, 100, 105, or 110 ng/ml). In some embodiments, the TGF-P ligand (e.g., activin A) has a concentration of about 8-12 ng/ml (e.g., 8, 9, 10, 11, or 12 ng/ml).

In some embodiments, the medium of an in vitro composition described herein further comprises an inhibitor of PI3K/Akt/mTOR signaling. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling may be selected from, but is not limited to, one or more of: GSK-690693, IPI-3063, AZD8055, Omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, Nemiralisib, BYL719, FT113, or Apitolisib, or any analog or derivative thereof. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling is GSK-690693 or an analog or derivative thereof. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693, or an analog or a derivative thereof) has a concentration of about 0.01-1 pM (e.g., 0.01-1, 0.01-0.8, 0.01-0.6, 0.01-0.4, 0.01-0.2, 0.01-0.1, 0.05-1, 0.05-0.8, 0.05-0.6, 0.05-0.4, 0.05-0.2, 0.05-0.1, 0.1-1, 0.1-0.8, 0.1-0.6, 0.1-0.4, 0.1-0.2, 0.2-1, 0.2-0.8, 0.2-0.5, 0.2-0.4, 0.4-1, 0.4-0.8, 0.4-0.6, 0.6-1, 0.6- 0.8, or 0.8-1 pM). In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693, or an analog or a derivative thereof) has a concentration of about 0.01-1 pM, 0.02-0.8 pM, 0.05-0.5 pM, 0.06-0.2 pM, 0.07-0.15 pM, or 0.08-0.12 pM. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693, or an analog or a derivative thereof) has a concentration of about 0.1 pM.

In some embodiments, the medium of an in vitro composition described herein further comprises a Wnt signaling pathway activator. In some embodiments, the Wnt signaling pathway activator may be a glycogen synthase kinase 3 (GSK3) inhibitor. In some embodiments, the glycogen synthase kinase 3 (GSK3) inhibitor is CHIR99021. In some embodiments, the Wnt signaling pathway activator (e.g., CHIR99021) has a concentration of 0.1-50 pM (e.g., 0.1-50, 0.1-25, 0.1-10, 0.1-5, 0.1-4, 0.1-3, 0.1-2, 0.1-1, 0.1-0.5, 0.5-50, 0.5-25, 0.5-10, 0.5-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1, 1-50, 1-25, 1-10, 1-5, 1-4, 1-3, 1-2, 2-50, 2-25, 2-10, 2-5, 2-4, 2-3, 3-50, 3-25, 3-10, 3-5, 3-4, 4-50, 4-25, 4-10, 4-5, 5-50, 5-25, 5-10, 10-50, 10-25, 25-50 pM). In some embodiments, the Wnt signaling pathway activator (e.g., CHIR99021) has a concentration of 2-4 pM (e.g., 2, 3, or 4 pM).

In some embodiments, the medium of an in vitro composition described herein further comprises a water-soluble synthetic polymer. In some embodiments, the water- soluble synthetic polymer is polyvinyl alcohol (PVA), poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide, optionally wherein the water-soluble synthetic polymer is polyvinyl alcohol. In some embodiments, the water water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the culture medium. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% (w/v), 0.01% (w/v), 0.05% (w/v), 0.1% (w/v), 0.15% (w/v), 0.2% (w/v), 0.25% (w/v), 0.3% (w/v), 0.35% (w/v), to 0.4% (w/v), 0.45% (w/v), or 0.5% (w/v) of the medium. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is at most 85% (e.g., 75%- 80%) hydrolyzed. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is about 80% hydrolyzed.

In some embodiments, an in vitro composition described herein further comprises definitive endoderm cells.

In some embodiments, the pluripotent stem cells of an in vitro composition described herein are embryonic stem cells. In some embodiments, the pluripotent stem cells of an in vitro composition described herein are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells of an in vitro composition described herein are human pluripotent stem cells. In some embodiments, the pluripotent stem cells are ABO blood group type O. In some embodiments, the pluripotent stem cells are genetically modified such that the cell is ABO blood group type O. In some embodiments, the pluripotent stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and/or HLA-DR, relative to cells that are not genetically modified. In some embodiments, the pluripotent stem cells have increased expression of one or more of CD47, PDL1, HLA-G, CD46, CD55, CD59 and/or CTLA, relative to cells that are not genetically modified.

Methods of producing pancreatic islet cells

In aspects, the present disclosure relates to compositions and methods of generating endocrine cells from pancreatic progenitor cells or precursors. Certain exemplary detailed protocols of generating endocrine cells to provide at least one SC-P cell are described in U.S. Patent Application Publication No. US20150240212, US20150218522, US 20200332262, US 20210198632, US 20220090020, US 2021- 0238553, US Patent 10,030,229; US Patent 10,443,042; and published application WO2022147056, each of which is herein incorporated by reference in its entirety. In some embodiments, a method of generating a population of endocrine cells leads to increased percentage of pancreatic a and/or 5 cells and decreased percentage of pancreatic EC cells when generating pancreatic P cells. In some embodiments, a method described herein may be used to obtain an enriched population of a cells. In some embodiments, a method described herein may be used to obtain an enriched population of P cells. In some embodiments, a method described herein may be used to obtain an enriched population of a cells and P cells. In some embodiments, a method described herein may be used to obtain an increased yield of pancreatic endocrine cells.

The differentiation of hPSC cells to hormone-expressing pancreatic endocrine cells may be conducted by transitioning hPSC cells through major stages of embryonic development; differentiation to mesendoderm and definitive endoderm, establishment of the primitive gut endoderm, patterning of the posterior foregut, and specification and maturation of pancreatic endoderm and endocrine precursors. Through these stages, hPSC cells can obtain pancreatic endocrine phenotype and ability of glucose responsive insulin secretion in vitro.

Generally, the at least one pancreatic SC-a, SC-P and/or SC-5 cell or precursor thereof, e.g., pancreatic progenitors produced according to the methods disclosed herein can comprise a mixture or combination of different cells, e.g., for example a mixture of cells such as a PDX1 -positive pancreatic progenitors, pancreatic progenitors co-expressing PDX1 and NKX6.1, a Ngn3-positive endocrine progenitor cell, an insulin-positive endocrine cell (e.g., NKX6.1 -positive, ISLl-positive cells, or P-like cells), and/or other pluripotent or stem cells.

The at least one pancreatic a, P and/or 5 cell or precursor thereof can be produced according to any suitable culturing protocol to differentiate a stem cell or pluripotent cell to a desired stage of differentiation. In some embodiments, the at least one pancreatic a, P and/or 5 cell or the precursor thereof are produced by culturing at least one pluripotent cell for a period of time and under conditions suitable for the at least one pluripotent cell to differentiate into the at least one pancreatic a, P and/or 5 cell or the precursor thereof.

In some embodiments, the at least one pancreatic a, P and/or 5 cell or precursor thereof is a substantially pure population of pancreatic a, P and/or 5 cells or precursors thereof. In some embodiments, a population of pancreatic a, P and/or 5 cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, a population pancreatic a, P and/or 5 cells or precursors thereof are substantially free or devoid of embryonic stem cells or pluripotent cells or iPS cells. In some embodiments, a method described herein produces a population of cells comprising pancreatic a, P and/or 5 cells at a ratio that resembles that of a natural pancreatic islet.

In some embodiments, a method described herein comprises: (a) culturing a first population of cells comprising pluripotent stem cells in a first medium supplemented with additional amino acids (e.g., aspartate, glycine, and/or serine) for a period of time to obtain a second population of cells; and (b) culturing the second population of cells in a second medium supplemented with additional amino acids (e.g., aspartate, glycine, and/or serine) for a period of time to obtain a third population of cells comprising definitive endoderm cells, wherein the second medium does not comprise a Wnt signaling activator. In some embodiments, a method described herein further comprises differentiating the definitive endoderm cells into pancreatic islet cells.

In some embodiments, in a method described herein, the first medium and/or the second medium further comprises a TGF-P ligand (e.g., activin A). In some embodiments, the first medium further comprises a Wnt signaling pathway activator (e.g., a glycogen synthase kinase 3 (GSK3) inhibitor such as CHIR99021). In some embodiments, the first medium and/or the second medium further comprises an inhibitor of PI3K/Akt/mT0R signaling (e.g., GSK-690693). In some embodiments, the first medium and/or the second medium further comprises a water-soluble synthetic polymer (e.g., PVA). In some embodiments, the first medium is supplemented with additional metabolites such as amino acids (e.g., aspartate, glycine, and/or serine) and further comprises a TGF-P ligand (e.g., activin A) and a Wnt signaling pathway activator (e.g., a glycogen synthase kinase 3 (GSK3) inhibitor such as CHIR99021), and optionally further comprises an inhibitor of PI3K/Akt/mT0R signaling (e.g., GSK-690693) and/or a water-soluble synthetic polymer (e.g., PVA). In some embodiments, the second medium is supplemented with additional amino acids (e.g., aspartate, glycine, and/or serine) and further comprises a TGF-P ligand (e.g., activin A), and optionally further comprises an inhibitor of PI3K/Akt/mT0R signaling (e.g., GSK-690693) and/or a water-soluble synthetic polymer (e.g., PVA), and does not comprise a Wnt signaling pathway activator.

In some embodiments, in a method described herein, the first medium and/or second medium comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM); (ii) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM); and/or (iii) serine at a concentration of higher than 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, in a method described herein, the first medium and/or second medium comprises aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM). In some embodiments, the first medium and/or second medium comprises glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM). In some embodiments, the first medium and/or second medium comprises serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, in a method described herein, the first medium and/or second medium comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM); and (ii) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM). In some embodiments, the first medium and/or second medium comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM), and (ii) serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM). In some embodiments, the first medium and/or second medium comprises (i) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM); and (ii) serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, in a method described herein, the first medium and/or second medium comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM); (ii) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM); and (iii) serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, in a method described herein, the first medium and/or second medium comprises aspartate, wherein the aspartate has a concentration of about 100-1000 pM (e.g., 100-1000, 100-800, 100-500, 100-400, 100-300, 100-250, 100-220, 100-210, 100-200, 100-190, 100-160, 100-120, 120-1000, 120-800, 120-500, 120-400, 120-300, 120-250, 120-220, 120-210, 120-200, 120-190, 120-160, 160-1000, 160-800, 160-500, 160-400, 160-300, 160-250, 160-220, 160-210, 160-200, 160-190, 190-1000, 190-800, 190-500, 190-400, 190-300, 190-250, 190-220, 190-210, 190-200, 200-1000, 200-800, 200-500, 200-400, 200-300, 200-250, 200-220, 200-210, 210-1000, 210-800, 210-500, 210-400, 210-300, 210-250, 210-220, 220-1000, 220-800, 220-500, 220-400, 220-300, 220-250, 250-1000, 250-800, 250-500, 250-400, 250-300, 300-1000, 300-800, 300-500, 300-400, 400-1000, 400-800, 400-500, 500-1000, 500-800, 800-1000 pM). In some embodiments, the aspartate has a concentration of about 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM. In particular embodiments, the aspartate has a concentration of 190-210 pM. In some embodiments, the aspartate has a concentration of about 100, 120, 160, 190, 200, 210, 220, 250, 300, 400, 500, 800, 1000 pM. In some embodiments, the aspartate has a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10 or more than 100 pM). In some embodiments, the aspartate has a concentration of about 200 pM.

In some embodiments, in a method described herein, the first medium and/or second medium comprises glycine, wherein the glycine has a concentration of about 30- 600 pM (e.g., 30-600, 30-500, 30-400, 30-350, 30-320, 30-300, 30-280, 30-200, 30-150, 30-100, 30-80, 30-40, 40-600, 40-500, 40-400, 40-350, 40-320, 40-300, 40-280, 40-200, 40-150, 40-100, 40-80, 80-600, 80-500, 80-400, 80-350, 80-320, 80-300, 80-280, 80-200, 80-150, 80-100, 100-600, 100-500, 100-400, 100-350, 100-320, 100-300, 100-280, 100- 200, 100-150, 150-600, 150-500, 150-400, 150-350, 150-320, 150-300, 150-280, 150-200, 200-600, 200-500, 200-400, 200-350, 200-320, 200-300, 200-280, 280-600, 280-500, 280- 400, 280-350, 280-320, 280-300, 300-600, 300-500, 300-400, 300-350, 300-320, 320-600, 320-500, 320-400, 320-350, 350-600, 350-500, 350-400, 400-600, 400-500, 500-600 pM). In some embodiments, the glycine has a concentration of about 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280- 320, or 150-350 pM. In particular embodiments, the glycine has a concentration of 280- 320 pM. In some embodiments, the glycine has a concentration of about 30, 40, 80, 100, 150, 200, 280, 300, 320, 350, 400, 500, 600 pM. In some embodiments, the glycine has a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10 or more than 30 pM). In some embodiments, the glycine has a concentration of about 300 pM.

In some embodiments, in a method described herein, the first medium and/or second medium comprises serine, wherein the serine has a concentration of about 285- 5000 pM (e.g., 285-5000, 285-4000, 285-3000, 285-2000, 285-1425, 285-1000, 285-800, 285-650, 285-620, 285-600, 285-585, 285-570, 285-550, 285-500, 285-400, 285-320, 320- 5000, 320-4000, 320-3000, 320-2000, 320-1425, 320-1000, 320-800, 320-650, 320-620, 320-600, 320-585, 320-570, 320-550, 320-500, 320-400, 400-5000, 400-4000, 400-3000, 400-2000, 400-1425, 400-1000, 400-800, 400-650, 400-620, 400-600, 400-585, 400-570, 400-550, 400-500, 500-5000, 500-4000, 500-3000, 500-2000, 500-1425, 500-1000, SOO- SOO, 500-650, 500-620, 500-600, 500-585, 500-570, 500-550, 550-5000, 550-4000, 550- 3000, 550-2000, 550-1425, 550-1000, 550-800, 550-650, 550-620, 550-600, 550-585, 550-570, 570-5000, 570-4000, 570-3000, 570-2000, 570-1425, 570-1000, 570-800, 570- 650, 570-620, 570-600, 570-585, 585-5000, 585-4000, 585-3000, 585-2000, 585-1425, 585-1000, 585-800, 585-650, 585-620, 585-600, 600-5000, 600-4000, 600-3000, 600- 2000, 600-1425, 600-1000, 600-800, 600-650, 600-620, 620-5000, 620-4000, 620-3000, 620-2000, 620-1425, 620-1000, 620-800, 620-650, 650-5000, 650-4000, 650-3000, 650- 2000, 650-1425, 650-1000, 650-800, 800-5000, 800-4000, 800-3000, 800-2000, SOO- 1425, 800-1000, 1000-5000, 1000-4000, 1000-3000, 1000-2000, 1000-1425, 1425-5000, 1425-4000, 1425-3000, 1425-2000, 2000-5000, 2000-4000, 2000-3000, 3000-5000, 3000- 4000, 4000-5000 pM). In some embodiments, the serine has a concentration of about 320- 5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500- 400, 320- 1425, 550-650, or 570-620 pM. In particular embodiments, the serine has a concentration of 570-620 pM. In some embodiments, the serine has a concentration of about 285, 320, 400, 500, 550, 570, 585, 600, 620, 650, 800, 1000, 1425, 2000, 3000, 4000, 5000 pM. In some embodiments, the medium comprises serine of a concentration at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10 or more than 285 pM). In some embodiments, the serine has a concentration about 585 pM.

In some embodiments, in a method described herein, the first medium and/or second medium comprises aspartate and glycine, wherein the aspartate has a concentration of about 100-1000 pM (e.g., 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM) and wherein the glycine has a concentration of about 30-600 pM (e.g., 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM). In some embodiments, the aspartate has a concentration of about 200 pM and the glycine has a concentration of about 300 pM. In some embodiments, in a method described herein, the first medium and/or second medium comprises aspartate and serine, wherein the aspartate has a concentration of about 100-1000 pM (e.g., 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM) and the serine has a concentration of about 285-5000 pM (320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500- 2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550-650, or 570-620 pM). In some embodiments, the aspartate has a concentration of about 200 pM and the serine has a concentration of about 585 pM.

In some embodiments, in a method described herein, the first medium and/or second medium comprises glycine and serine, wherein the glycine has a concentration of about 30-600 pM (e.g., 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, SOO- SOO, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM) and the serine has a concentration of about 285-5000 pM (320-5000, 320-4000, 320-3000, 320-2000, 320- 1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550-650, or 570-620 pM). In some embodiments, the glycine has a concentration of about 300 pM and the serine has a concentration of about 585 pM.

In some embodiments, in a method described herein, the first medium and/or second medium comprises aspartate, glycine, and serine, wherein the aspartate has a concentration of about 100-1000 pM (e.g., 120-1000, 120-800, 120- 500, 120-400, 120- 300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM), the glycine has a concentration of about 30-600 pM (e.g., 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM), and the serine has a concentration of about 285-5000 pM (320-5000, 320- 4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550-650, or 570-620 pM). In one embodiment, the aspartate has concentration of about 200 pM, the glycine has a concentration of about 300 pM, and the serine has a concentration of about 585 pM. In some embodiments, in a method described herein, the first medium and/or second medium further comprises a TGF-P ligand (e.g., activin A). In some embodiments, the TGF-P ligand (e.g., activin A) has a concentration of about 1-200 ng/ml (e.g., 1-200, 1- 150, 1-125, 1-110, 1-100, 1-90, 1-75, 1-50, 1-25, 1-15, 1-12, 1-10, 1-8, 1-5, 5-200, 5-150, 5-125, 5-110, 5-100, 5-90, 5-75, 5-50, 5-25, 5-15, 5-12, 5-10, 5-8, 8-200, 8-150, 8-125, 8- 110, 8-100, 8-90, 8-75, 8-50, 8-25, 8-15, 8-12, 8-10, 10-200, 10-150, 10-125, 10-110, 10- 100, 10-90, 10-75, 10-50, 10-25, 10-15, 10-12, 12-200, 12-150, 12-125, 12-110, 12-100, 12-90, 12-75, 12-50, 12-25, 12-15, 15-200, 15-150, 15-125, 15-110, 15-100, 15-90, 15-75, 15-50, 15-25, 25-200, 25-150, 25-125, 25-110, 25-100, 25-90, 25-75, 25-50, 50-200, 50- 150, 50-125, 50-110, 50-100, 50-90, 50-75, 75-200, 75-150, 75-125, 75-110, 75-100, 75- 90, 90-200, 90-150, 90-125, 90-110, 90-100, 100-200, 100-150, 100-125, 100-110, 110- 200, 110-250, 110-125, 125-200, 125-150, or 150-200 ng/ml). In some embodiments, the TGF-P ligand (e.g., activin A) has a concentration of about 1-50, 1-25, 5-50, 5-25, 5-15, 8- 12, 10-1000, 10-500, 10-250, 10-125, 75-1000, 75-500, 75-250, 75-125, or 90-110 ng/ml. In some embodiments, the TGF-P ligand (e.g., activin A) has a concentration of about 90- 110 ng/ml (e.g., 90, 95, 100, 105, or 110 ng/ml). In some embodiments, the TGF-P ligand (e.g., activin A) has a concentration of about 8-12 ng/ml (e.g., 8, 9, 10, 11, or 12 ng/ml).

In some embodiments, in a method described herein, the first medium and/or second medium further comprises an inhibitor of PI3K/Akt/mTOR signaling. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling may be selected from, but is not limited to, one or more of GSK-690693, IPI-3063, AZD8055, Omipalisib, GNE-477, VS- 5584, BYL319, YM201636, PI4KIIIbeta-IN-10, Nemiralisib, BYL719, FT113, or Apitolisib, or any analog or derivative thereof. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling is GSK-690693 or an analog or derivative thereof. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693, or an analog or a derivative thereof) has a concentration of about 0.01-1 pM (e.g., 0.01-1, 0.01-0.8, 0.01-0.6, 0.01-0.4, 0.01-0.2, 0.01-0.1, 0.05-1, 0.05-0.8, 0.05-0.6, 0.05-0.4, 0.05-0.2, 0.05- 0.1, 0.1-1, 0.1-0.8, 0.1-0.6, 0.1-0.4, 0.1-0.2, 0.2-1, 0.2-0.8, 0.2-0.5, 0.2-0.4, 0.4-1, 0.4-0.8, 0.4-0.6, 0.6-1, 0.6-0.8, or 0.8-1 pM). In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693, or an analog or a derivative thereof) has a concentration of about 0.01-1 pM, 0.02-0.8 pM, 0.05-0.5 pM, 0.06-0.2 pM, 0.07-0.15 pM, or 0.08-0.12 pM. In some embodiments, the inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693, or an analog or a derivative thereof) has a concentration of about 0.1 pM. In some embodiments, in a method described herein, the first medium further comprises a Wnt signaling pathway activator. In some embodiments, the Wnt signaling pathway activator may be a glycogen synthase kinase 3 (GSK3) inhibitor. In some embodiments, the glycogen synthase kinase 3 (GSK3) inhibitor is CHIR99021. In some embodiments, the Wnt signaling pathway activator (e.g., CHIR99021) has a concentration of 0.1-50 pM (e.g., 0.1-50, 0.1-25, 0.1-10, 0.1-5, 0.1-4, 0.1-3, 0.1-2, 0.1-1, 0.1-0.5, 0.5-50, 0.5-25, 0.5-10, 0.5-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1, 1-50, 1-25, 1-10, 1-5, 1-4, 1-3, 1-2, 2-50, 2-25, 2-10, 2-5, 2-4, 2-3, 3-50, 3-25, 3-10, 3-5, 3-4, 4-50, 4-25, 4-10, 4-5, 5-50, 5-25, 5- 10, 10-50, 10-25, 25-50 pM). In some embodiments, the Wnt signaling pathway activator (e.g., CHIR99021) has a concentration of 2-4 pM (e.g., 2, 3, or 4 pM).

In some embodiments, in a method described herein, the first medium and/or second medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N- isopropylacrylamide), or polyacrylamide, optionally wherein the water-soluble synthetic polymer is polyvinyl alcohol. In some embodiments, the water water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the culture medium. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% (w/v), 0.01% (w/v), 0.05% (w/v), 0.1% (w/v), 0.15% (w/v), 0.2% (w/v), 0.25% (w/v), 0.3% (w/v), 0.35% (w/v), to 0.4% (w/v), 0.45% (w/v), or 0.5% (w/v) of the medium. In some embodiments, the water- soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is at most 85% (e.g., 75%-80%) hydrolyzed. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is about 80% hydrolyzed.

In some embodiments, in a method described herein, the first population of cells is cultured in the first medium for a period of about 18-48 hours (e.g., about 18-48 hours, 18- 42 hours, 18-36 hours, 18-30 hours, 18-24 hours, 24-48 hours, 24-42 hours, 24-36 hours, 24-30 hours, 30-48 hours, 30-42 hours, 30-36 hours, 36-48 hours, 36-42 hours, or 42-48 hours). In some embodiments, the first population of cells is cultured in the first medium for a period of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours. In some embodiments, the first population of cells is cultured in the first medium for a period of about 24 hours. In some embodiments, culturing the first population of cells in the first media for a contacting period described herein (e.g., 24 hours) results in a second population of cells.

In some embodiments, a method described herein further comprises culturing the second population of cells with the second medium for a period of 36-72 hours (e.g., 36- 72 hours, 36-66 hours, 36-60 hours, 36-54 hours, 36-48 hours, 36-42 hours, 42-72 hours, 42-66 hours, 42-60 hours, 42-54 hours, 42-48 hours, 48-72 hours, 48-66 hours, 48-60 hours, 48-54 hours, 54-72 hours, 54-66 hours, 54-60 hours, 60-72 hours, 60-66 hours, or 66-72 hours). In some embodiments, the second population of cells is cultured in the second medium for a period of about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours. In some embodiments, the second population of cells is cultured in the second medium for a period of about 48 hours. In some embodiments, culturing the second population of cells in the second media for a contacting period described herein (e.g., 24 hours) results in a third population of cells. In some embodiments, the third population of cells comprise definitive endoderm cells. In some embodiments, the third population of cells further comprise pluripotent stem cells and/or cells that are at a differentiation stage of between pluripotent stem cells and definitive endoderm cells.

In some embodiments, the pluripotent stem cells used in a method described herein are embryonic stem cells. In some embodiments, the pluripotent stem cells used in a method described herein are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells used in a method described herein are human pluripotent stem cells. In some embodiments, the pluripotent stem cells are ABO blood group type O. In some embodiments, the pluripotent stem cells are genetically modified such that the cell is ABO blood group type O. In some embodiments, the pluripotent stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, relative to cells that are not genetically modified. In some embodiments, the pluripotent stem cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified.

In some embodiments, a method described herein further comprises differentiating (e.g., using any methods described herein or known in the art) the definitive endoderm cells to pancreatic endocrine cells (e.g., P cells, a cells, and 5 cells). Cell types during pancreatic differentiation

Aspects of the present disclosure provide cell types of the pancreatic lineage obtained during differentiation of stem cells to generate pancreatic islet cells. Such cells include any cell that is capable of differentiating into a pancreatic islet cell, including for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell, or endocrine progenitor cell, when cultured under conditions suitable for differentiating the precursor cell into the pancreatic islet cell.

Stem Cells

“Stem cell” refers to a cell (e.g., plant stem cell, vertebrate stem cell) that has the ability both to self-renew and to generate a differentiated cell type (Morrison et al. (1997) Cell 88:287-298). In the context of cell ontogeny, the adjective “differentiated,” or “differentiating” is a relative term. A “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which in turn can differentiate into cells that are further restricted (e.g., neuron progenitors), which can differentiate into end-stage cells (i.e., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.), which play a characteristic role in a certain tissue type, and can or cannot retain the capacity to proliferate further. Stem cells can be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells can also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny. In an embodiment, the host cell is an adult stem cell, a somatic stem cell, a non- embryonic stem cell, an embryonic stem cell, hematopoietic stem cell, an include pluripotent stem cells, and a trophoblast stem cell.

Stem cells of interest include pluripotent stem cells (PSCs). The term “pluripotent stem cell” or “PSC” is used herein to mean a stem cell capable of producing all cell types of the organism. Therefore, a PSC can give rise to cells of all germ layers of the organism (e.g., the endoderm, mesoderm, and ectoderm of a vertebrate). Pluripotent cells are capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism. Pluripotent stem cells of plants are capable of giving rise to all cell types of the plant (e.g., cells of the root, stem, leaves, etc.). PSCs of animals can be derived in a number of different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of an embryo (Thomson et. al, Science. 1998 Nov. 6; 282(5391): 1145-7) whereas induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi et. al, Cell. 2007 Nov. 30; 13 1(5):861- 72; Takahashi et. al, Nat Protoc. 2007; 2(12):3081-9; Yu et. al, Science. 2007 Dec. 21; 318(5858): 1917-20. Epub 2007 Nov. 20). Because the term PSC refers to pluripotent stem cells regardless of their derivation, the term PSC encompasses the terms ESC and iPSC, as well as the term embryonic germ stem cells (EGSC), which are another example of a PSC. PSCs can be in the form of an established cell line, they can be obtained directly from primary embryonic tissue, or they can be derived from a somatic cell.

By “embryonic stem cell” (ESC) is meant a PSC that is isolated from an embryo, typically from the inner cell mass of the blastocyst. ESC lines are listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-Ol, hESBGN-02, hESBGN-03, hESBGN- 04 (BresaGen, Inc ); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz- hESl (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and Hl, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as Rhesus stem cells and marmoset stem cells. The stem cells can be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. (Thomson et al. (1998) Science 282: 1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254;

Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998). In culture, ESCs typically grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nucleoli. In addition, ESCs express S SEA-3, S SEA-4, TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not S SEA-1 . Examples of methods of generating and characterizing ESCs may be found in, for example, U.S. Pat. No. 7,029,913, U.S. Pat. No. 5,843,780, and U.S. Pat. No. 6,200,806, each of which is incorporated herein by its entirety. Methods for proliferating hESCs in the undifferentiated form are described in WO 99/20741, WO 01/51616, and WO 03/020920, each of which is incorporated herein by its entirety.

By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EG cell,” it is meant a PSC that is derived from germ cells and/or germ cell progenitors, e.g., primordial germ cells, i.e. those that can become sperm and eggs. Embryonic germ cells (EG cells) are thought to have properties similar to embryonic stem cells as described above. Examples of methods of generating and characterizing EG cells may be found in, for example, U.S. Pat. No. 7, 153,684; Matsui, Y., et al., (1992) Cell 70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95: 13726; and Koshimizu, U., et al. (1996) Development, 122: 1235, each of which are incorporated herein by its entirety. By “induced pluripotent stem cell” or “iPSC,” it is meant a PSC that is derived from a cell that is not a PSC (i.e., from a cell this is differentiated relative to a PSC). iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42. Examples of methods of generating and characterizing iPSCs can be found in, for example, Patent Publication Nos.

US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, each of which are incorporated herein by its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells.

By “somatic cell,” it is meant any cell in an organism that, in the absence of experimental manipulation, does not ordinarily give rise to all types of cells in an organism. In other words, somatic cells are cells that have differentiated sufficiently that they do not naturally generate cells of all three germ layers of the body, i.e., ectoderm, mesoderm and endoderm. For example, somatic cells can include both neurons and neural progenitors, the latter of which is able to naturally give rise to all or some cell types of the central nervous system but cannot give rise to cells of the mesoderm or endoderm lineages.

In certain examples, the stem cells can be undifferentiated (e.g., a cell not committed to a specific lineage) prior to exposure to at least one cell maturation factor according to the methods as disclosed herein, whereas in other examples it may be desirable to differentiate the stem cells to one or more intermediate cell types prior to exposure of the at least one cell maturation factor (s) described herein. For example, the stems cells may display morphological, biological or physical characteristics of undifferentiated cells that can be used to distinguish them from differentiated cells of embryo or adult origin. In some examples, undifferentiated cells may appear in the two dimensions of a microscopic view in colonies of cells with high nuclear/cytoplasmic ratios and prominent nucleoli. The stem cells may be themselves (for example, without substantially any undifferentiated cells being present) or may be used in the presence of differentiated cells. In certain examples, the stem cells may be cultured in the presence of suitable nutrients and optionally other cells such that the stem cells can grow and optionally differentiate. For example, embryonic fibroblasts or fibroblast-like cells may be present in the culture to assist in the growth of the stem cells. The fibroblast may be present during one stage of stem cell growth but not necessarily at all stages. For example, the fibroblast may be added to stem cell cultures in a first culturing stage and not added to the stem cell cultures in one or more subsequent culturing stages.

Stem cells used in all aspects of the present invention can be any cells derived from any kind of tissue (for example embryonic tissue such as fetal or pre-fetal tissue, or adult tissue), which stem cells have the characteristic of being capable under appropriate conditions of producing progeny of different cell types, e.g., derivatives of all of at least one of the 3 germinal layers (endoderm, mesoderm, and ectoderm). These cell types may be provided in the form of an established cell line, or they may be obtained directly from primary embryonic tissue and used immediately for differentiation. Included are cells listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-Ol, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc ); HES-1, HES-2, HES-3, HES-4, HES-5, HES- 6 (ES Cell International); Miz-hESl (MizMedi Hospital -Seoul National University); HSF- 1, FISF-6 (University of California at San Francisco); and Hl, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically-induced differentiation into mature, insulin positive cells did not involve destroying a human embryo.

In another embodiment, the stem cells can be isolated from tissue including solid tissue. In some embodiments, the tissue is skin, fat tissue (e.g., adipose tissue), muscle tissue, heart or cardiac tissue. In other embodiments, the tissue is for example but not limited to, umbilical cord blood, placenta, bone marrow, or chondral. Stem cells of interest also include embryonic cells of various types, exemplified by human embryonic stem (hES) cells, described by Thomson et al, (1998) Science 282: 1145; embryonic stem cells from other primates, such as Rhesus stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998). Also of interest are lineage committed stem cells, such as mesodermal stem cells and other early cardiogenic cells (see Reyes et al, (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2):205-16; etc.). The stem cells may be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g., mice, rats, hamster, primate, etc. In some embodiments, a human embryo was not destroyed for the source of pluripotent cell used on the methods and compositions as disclosed herein.

A mixture of cells from a suitable source of endothelial, muscle, and/or neural stem cells can be harvested from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (i.e., recruited), may be removed from a subject. In an embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic or differentiated cells. In such an embodiment, the de-differentiated stem cells can be for example, but not limited to, neoplastic cells, tumor cells and cancer cells or alternatively induced reprogrammed cells such as induced pluripotent stem cells or iPS cells.

In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are induced pluripotent stem cells. In some embodiments, the stem cells used in a method described herein are human stem cells. In some embodiments, the stem cells are ABO blood group type O. In some embodiments, the stem cells are genetically modified such that the cell is ABO blood group type O. In some embodiments, the stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, relative to cells that are not genetically modified. In some embodiments, the stem cells have increased expression of one or more of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified. Definitive Endoderm Cells

The definitive endoderm can be generated in vivo from the inner cell mass by the process of gastrulation of embryogenesis, in which epiblast cells are instructed to form the three germ layers. Definitive endoderm can give rise to diverse cells and tissues that contribute to vital organs as the pancreatic P cells, liver hepatocytes, lung alveolar cells, thyroid, thymus, and the epithelial lining of the alimentary and respiratory tract. It is different from the primitive endoderm of extraembryonic tissues, which can give rise to the visceral and parietal endoderm. The definitive endoderm derived from ES cells is theoretically capable of becoming any endoderm derivatives.

Precise patterning of anterior-posterior axis of the definitive endoderm can eventually form the primitive gut tube. The definitive endoderm-derived primitive gut tube induces the pharynx, esophagus, stomach, duodenum, small and large intestine along the anterior-posterior axis as well as associated organs, including pancreas, lung, thyroid, thymus, parathyroid, and liver. The anterior portion of the foregut of the primitive gut tube becomes lung, thyroid, esophagus, and stomach. The pancreas, liver, and duodenum originate from the posterior portion of the foregut. The midgut and hindgut of primitive gut tube gives rise to the small and large intestine. The anterior foregut expresses developmental markers, NK2 homeobox 1 (NKX2-1) and SRY (sex determining region Y)-box 2 (SOX2); the posterior foregut expresses hematopoietically expressed homeobox (HHEX), pancreatic and duodenal homeobox 1 (PDX1), one cut homeobox 1 (0NECUT1, known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A); and the midgut/hindgut expresses caudal type homeobox 1 (CDX1), caudal type homeobox 2 (CDX2), and motor neuron and pancreas homeobox 1 (MNX1) (3, 19, 20).

As described herein definitive endoderm cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, pluripotent stem cells, e.g., iPSCs or hESCs, are differentiated to endoderm cells. In some aspects, the endoderm cells (stage 1) are further differentiated, e.g., to primitive gut tube cells (stage 2), PDXl-positive pancreatic progenitor cells (stage 3), NKX6.1 -positive pancreatic progenitor cells (stage 4), or Ngn3 -positive endocrine progenitor cells or insulin-positive endocrine cells (stage 5), followed by induction or maturation to SC-P cells (stage 6). In some embodiments, definitive endoderm cells can be obtained by differentiating at least some pluripotent cells in a population into definitive endoderm cells, e.g., by contacting a population of pluripotent cells with i) at least one growth factor from the TGF-P superfamily, and ii) a WNT signaling pathway activator, to induce the differentiation of at least some of the pluripotent cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

In some embodiments, definitive endoderm cells can be obtained by differentiating at least some pluripotent cells in a population into definitive endoderm cells, e.g., by contacting a population of pluripotent cells with a medium supplemented with additional amino acids (e.g., aspartate, glycine, and/or serine). The medium may further comprise one or more of: i) at least one growth factor from the TGF-P superfamily, ii) a WNT signaling pathway activator, and (iii) an inhibitor of PI3K/Akt/mT0R signaling, to induce the differentiation of at least some of the pluripotent cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

In some embodiments, the medium supplemented with additional amino acids comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM); (ii) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM); and/or (iii) serine at a concentration of higher than 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, the medium supplemented with additional amino acids comprises aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM). In some embodiments, the medium supplemented with additional amino acids comprises glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM). In some embodiments, the medium supplemented with additional amino acids comprises serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, the medium supplemented with additional amino acids (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM); and (ii) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM). In some embodiments, the first medium and/or second medium comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM), and (ii) serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM). In some embodiments, the medium supplemented with additional amino acids comprises (i) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM); and (ii) serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, the medium supplemented with additional amino acids comprises (i) aspartate at a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 100 pM); (ii) glycine at a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 30 pM); and (iii) serine at a concentration of at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10, 20 pM higher or more than 285 pM).

In some embodiments, the medium supplemented with additional amino acids comprises aspartate, wherein the aspartate has a concentration of about 100-1000 pM (e.g., 100-1000, 100-800, 100-500, 100-400, 100-300, 100-250, 100-220, 100-210, 100- 200, 100-190, 100-160, 100-120, 120-1000, 120-800, 120-500, 120-400, 120-300, 120- 250, 120-220, 120-210, 120-200, 120-190, 120-160, 160-1000, 160-800, 160-500, 160-

400, 210-300, 210-250, 210-220, 220-1000, 220-800, 220-500, 220-400, 220-300, 220- 250, 250-1000, 250-800, 250-500, 250-400, 250-300, 300-1000, 300-800, 300-500, 300- 400, 400-1000, 400-800, 400-500, 500-1000, 500-800, 800-1000 pM). In some embodiments, the aspartate has a concentration of about 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM. In some embodiments, the aspartate has a concentration of about 100, 120, 160, 190, 200, 210, 220, 250, 300, 400, 500, 800, 1000 pM. In some embodiments, the aspartate has a concentration of at least 100 pM (e.g., at least 0.1, 0.5, 1, 5, 10 or more than 100 pM). In some embodiments, the aspartate has a concentration of about 200 pM.

In some embodiments, the medium supplemented with additional amino acids comprises glycine, wherein the glycine has a concentration of about 30-600 pM (e.g., 30- 600, 30-500, 30-400, 30-350, 30-320, 30-300, 30-280, 30-200, 30-150, 30-100, 30-80, 30- 40, 40-600, 40-500, 40-400, 40-350, 40-320, 40-300, 40-280, 40-200, 40-150, 40-100, 40- 80, 80-600, 80-500, 80-400, 80-350, 80-320, 80-300, 80-280, 80-200, 80-150, 80-100, 100-600, 100-500, 100-400, 100-350, 100-320, 100-300, 100-280, 100-200, 100-150, 150- 600, 150-500, 150-400, 150-350, 150-320, 150-300, 150-280, 150-200, 200-600, 200-500, 200-400, 200-350, 200-320, 200-300, 200-280, 280-600, 280-500, 280-400, 280-350, 280- 320, 280-300, 300-600, 300-500, 300-400, 300-350, 300-320, 320-600, 320-500, 320-400, 320-350, 350-600, 350-500, 350-400, 400-600, 400-500, 500-600 pM). In some embodiments, the glycine has a concentration of about 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM. In some embodiments, the glycine has a concentration of about 30, 40, 80, 100, 150, 200, 280, 300, 320, 350, 400, 500, 600 pM. In some embodiments, the glycine has a concentration of at least 30 pM (e.g., at least 0.1, 0.5, 1, 5, 10 or more than 30 pM). In some embodiments, the glycine has a concentration of about 300 pM.

In some embodiments, the medium supplemented with additional amino acids comprises serine, wherein the serine has a concentration of about 285-5000 pM (e.g., 285- 5000, 285-4000, 285-3000, 285-2000, 285-1425, 285-1000, 285-800, 285-650, 285-620, 285-600, 285-585, 285-570, 285-550, 285-500, 285-400, 285-320, 320-5000, 320-4000, 320-3000, 320-2000, 320-1425, 320-1000, 320-800, 320-650, 320-620, 320-600, 320-585, 320-570, 320-550, 320-500, 320-400, 400-5000, 400-4000, 400-3000, 400-2000, 400- 1425, 400-1000, 400-800, 400-650, 400-620, 400-600, 400-585, 400-570, 400-550, 400- 500, 500-5000, 500-4000, 500-3000, 500-2000, 500-1425, 500-1000, 500-800, 500-650, 500-620, 500-600, 500-585, 500-570, 500-550, 550-5000, 550-4000, 550-3000, 550-2000, 550-1425, 550-1000, 550-800, 550-650, 550-620, 550-600, 550-585, 550-570, 570-5000, 570-4000, 570-3000, 570-2000, 570-1425, 570-1000, 570-800, 570-650, 570-620, 570- 600, 570-585, 585-5000, 585-4000, 585-3000, 585-2000, 585-1425, 585-1000, 585-800, 585-650, 585-620, 585-600, 600-5000, 600-4000, 600-3000, 600-2000, 600-1425, 600- 1000, 600-800, 600-650, 600-620, 620-5000, 620-4000, 620-3000, 620-2000, 620-1425, 620-1000, 620-800, 620-650, 650-5000, 650-4000, 650-3000, 650-2000, 650-1425, 650- 1000, 650-800, 800-5000, 800-4000, 800-3000, 800-2000, 800-1425, 800-1000, 1000- 5000, 1000-4000, 1000-3000, 1000-2000, 1000-1425, 1425-5000, 1425-4000, 1425-3000, 1425-2000, 2000-5000, 2000-4000, 2000-3000, 3000-5000, 3000-4000, 4000-5000 pM). In some embodiments, the serine has a concentration of about 320-5000, 320-4000, 320- 3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550- 650, or 570-620 pM. In some embodiments, the serine has a concentration of about 285, 320, 400, 500, 550, 570, 585, 600, 620, 650, 800, 1000, 1425, 2000, 3000, 4000, 5000 pM. In some embodiments, the medium comprises serine of a concentration at least 285 pM (e.g., at least 0.1, 0.5, 1, 5, 10 or more than 285 pM). In some embodiments, the serine has a concentration about 585 pM.

In some embodiments, the medium supplemented with additional amino acids comprises aspartate and glycine, wherein the aspartate has a concentration of about 100- 1000 pM (e.g., 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160- 300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM) and wherein the glycine has a concentration of about 30-600 pM (e.g., 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200- 300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM). . In some embodiments, the aspartate has a concentration of about 200 pM and the glycine has a concentration of about 300 pM.

In some embodiments, the medium supplemented with additional amino acids comprises aspartate and serine, wherein the aspartate has a concentration of about 100- 1000 pM (e.g., 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160- 300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM) and the serine has a concentration of about 285-5000 pM (320-5000, 320-4000, 320-3000, 320-2000, 320- 1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550-650, or 570-620 pM). In some embodiments, the aspartate has a concentration of about 200 pM and the serine has a concentration of about 585 pM.

In some embodiments, the medium supplemented with additional amino acids comprises glycine and serine, wherein the glycine has a concentration of about 30-600 pM (e.g., 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400- 600, 400-600, 500-600, 280-320, or 150-350 pM) and the serine has a concentration of about 285-5000 pM (320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320- 600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550-650, or 570-620 pM). In some embodiments, the glycine has a concentration of about 300 pM and the serine has a concentration of about 585 pM. In some embodiments, the medium supplemented with additional amino acids comprises aspartate, glycine, and serine, wherein the aspartate has a concentration of about 100-1000 pM (e.g., 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM), the glycine has a concentration of about 30-600 pM (e.g., 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200- 300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM), and the serine has a concentration of about 285-5000 pM (320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500- 3000, 500-2000, 500-1000, 500-800, 500-600, 500-500, 500-400, 320- 1425, 550-650, or 570-620 pM). In one embodiment, the aspartate has concentration of about 200 pM, the glycine has a concentration of about 300 pM, and the serine has a concentration of about 585 pM.

Any growth factor from the TGF-P superfamily capable of inducing the pluripotent stem cells to differentiate into definitive endoderm cells (e.g., alone, or in combination with a WNT signaling pathway activator) can be used in the method provided herein. In some embodiments, the growth factor from the TGF-P superfamily comprises Activin A. In some embodiments, the growth factor from the TGF-P superfamily comprises growth differentiating factor 8 (GDF8). Any WNT signaling pathway activator capable of inducing the pluripotent stem cells to differentiate into definitive endoderm cells (e.g., alone, or in combination with a growth factor from the TGF-P superfamily) can be used in the method provided herein. In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator comprises Wnt3a recombinant protein.

In some embodiments, differentiating at least some pluripotent cells in a population into definitive endoderm cells is achieved by a process of contacting a population of pluripotent cells with i) Activin A, and ii) CHIR99021 for a suitable period of time, e.g., about 2 days, about 3 days, about 4 days, or about 5 days to induce the differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm. In some embodiments, the process comprises contacting a population of pluripotent cells with activin A and CHIR99021 for 1 day, and then with activin A (in the absence of CHTR.99021) for a further 1 or 2 days. In some embodiments, on each of the days, the cells are further in contact with an inhibitor of PI3K/Akt/mT0R signaling.

In some examples, the method comprises differentiating pluripotent cells into definitive endoderm cells by contacting a population of pluripotent cells with a suitable concentration of the growth factor from the TGF-P superfamily (e.g., Activin A), such as, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some embodiments, the method comprises use of about 70-130 ng. ml, 80-120 ng/ml, or 90-110 ng/ml Activin A for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 100 ng/mL Activin A for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 200 ng/mL Activin A for differentiation of pluripotent cells into definitive endoderm cells.

In some examples, the method comprises differentiating pluripotent cells into definitive endoderm cells by contacting a population of pluripotent cells with a suitable concentration of the WNT signaling pathway activator (e.g., CHIR99021), such as, about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.5 pM, about 0.8 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 5 pM, about 8 pM, about 10 pM, about 12 pM, about 15 pM, about 20 pM, about 30 pM, about 50 pM, about 100 pM, or about 200 pM. In some embodiments, the method comprises use of about 1-5 pM or 2-4 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 2 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 3 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 5 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells.

In some embodiments, the method comprises use of about 1-5 pM or 2-4 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 2 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 3 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 5 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells.

In some examples, the method comprises differentiating pluripotent cells into definitive endoderm cells by contacting a population of pluripotent cells with a suitable concentration of the an inhibitor of PI3K/Akt/mT0R signaling, such as, about 0.01-1 pM (e.g., 0.01-1, 0.01-0.8, 0.01-0.6, 0.01-0.4, 0.01-0.2, 0.01-0.1, 0.05-1, 0.05-0.8, 0.05-0.6, 0.05-0.4, 0.05-0.2, 0.05-0.1, 0.1-1, 0.1-0.8, 0.1-0.6, 0.1-0.4, 0.1-0.2, 0.2-1, 0.2-0.8, 0.2- 0.5, 0.2-0.4, 0.4-1, 0.4-0.8, 0.4-0.6, 0.6-1, 0.6-0.8, or 0.8-1 pM). In some embodiments, the method comprises use of about 0.01-1 pM, 0.02-0.8 pM, 0.05-0.5 pM, 0.06-0.2 pM, 0.07-0.15 pM, or 0.08-0.12 pM of the inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK- 690693, or an analog or a derivative thereof) for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 0.1 pM of the inhibitor of PI3K/Akt/mTOR signaling (e.g., GSK-690693, or an analog or a derivative thereof) for differentiation of pluripotent cells into definitive endoderm cells.

In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some cases, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 80% hydrolyzed.

In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein expresses at least one marker selected from the group consisting of: Nodal, Tmprss2, Tmem30b, Stl4, Spink3, Sh3gl2, Ripk4, RablS, Npnt, Clic6, Cldn5, Cacnalb, Bnipl, Anxa4, Emb, FoxAl, Soxl7, and Rbm35a, wherein the expression of at least one marker is upregulated to by a statistically significant amount in the definitive endoderm cell relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein does not express by a statistically significant amount at least one marker selected the group consisting of: Gata4, SPARC, AFP and Dab2 relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein does not express by a statistically significant amount at least one marker selected the group consisting of: Zicl, Pax6, Flkl and CD31 relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein has a higher level of phosphorylation of Smad2 by a statistically significant amount relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein has the capacity to form gut tube in vivo. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein can differentiate into a cell with morphology characteristic of a gut cell, and wherein a cell with morphology characteristic of a gut cell expresses FoxA2 and/or Claudin6. In some embodiments, a definitive endoderm cell produced by the methods as disclosed herein can be further differentiated into a cell of endoderm origin.

In some embodiments, a population of pluripotent stem cells are cultured in the presence of at least one P cell differentiation factor prior to any differentiation or during the first stage of differentiation. One can use any pluripotent stem cell, such as a human pluripotent stem cell, or a human iPS cell or any of pluripotent stem cell as discussed herein or other suitable pluripotent stem cells. In some embodiments, a P cell differentiation factor as described herein can be present in the culture medium of a population of pluripotent stem cells or may be added in bolus or periodically during growth (e.g. replication or propagation) of the population of pluripotent stem cells. In certain examples, a population of pluripotent stem cells can be exposed to at least one P cell differentiation factor prior to any differentiation. In other examples, a population of pluripotent stem cells may be exposed to at least one P cell differentiation factor during the first stage of differentiation.

Primitive Gut Tube Cells

Aspects of the disclosure involve primitive gut tube cells. Primitive gut tube cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, definitive endoderm cells are differentiated to primitive gut tube cells. In some aspects, the primitive gut tube cells are further differentiated, e.g., to PDX1- positive pancreatic progenitor cells, NKX6.1 -positive pancreatic progenitor cells, Ngn3- positive endocrine progenitor cells, insulin-positive endocrine cells, followed by induction or maturation to SC-P cells. In some embodiments, primitive gut tube cells can be obtained by differentiating at least some definitive endoderm cells in a population into primitive gut tube cells, e.g., by contacting definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family, to induce the differentiation of at least some of the definitive endoderm cells into primitive gut tube cells, wherein the primitive gut tube cells express at least one marker characteristic of primitive gut tube cells.

Any growth factor from the FGF family capable of inducing definitive endoderm cells to differentiate into primitive gut tube cells (e.g., alone, or in combination with other factors) can be used in the method provided herein. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family comprises FGF2. In some embodiments, the at least one growth factor from the FGF family comprises FGF8B. In some embodiments, the at least one growth factor from the FGF family comprises FGF10. In some embodiments, the at least one growth factor from the FGF family comprises FGF21.

In some embodiments, primitive gut tube cells can be obtained by differentiating at least some definitive endoderm cells in a population into primitive gut tube cells, e.g., by contacting definitive endoderm cells with KGF for a certain period of time, e.g., about 1 day, about 2 days, about 3 days, or about 4 days, to induce the differentiation of at least some of the definitive endoderm cells into primitive gut tube cells.

In some embodiments, the method comprises differentiating definitive endoderm cells into primitive gut tube cells by contacting definitive endoderm cells with a suitable concentration of the growth factor from the FGF family (e.g., KGF), such as, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some embodiments, the method comprises use of about 20-80 ng/ml, 30-70 ng/ml, or 40-60 ng/mL KGF for differentiation of definitive endoderm cells into primitive gut tube cells. In some embodiments, the method comprises use of about 50 ng/mL KGF for differentiation of definitive endoderm cells into primitive gut tube cells. In some embodiments, the method comprises use of about 100 ng/mL KGF for differentiation of definitive endoderm cells into primitive gut tube cells. In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some cases, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 80% hydrolyzed.

PDXl-positive Pancreatic Progenitor Cells

Aspects of the disclosure involve PDXl-positive pancreatic progenitor cells. PDXl- positive pancreatic progenitor cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, primitive gut tube cells are differentiated to PDX1 -positive pancreatic progenitor cells. In some aspects, the PDXl-positive pancreatic progenitor cells are NKX6.1 negative, and can be further differentiated to, e.g., NKX6.1 -positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, followed by induction or maturation to SC-P cells.

In some aspects, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with one or more of i) at least one BMP signaling pathway inhibitor, ii) a growth factor from TGF-P superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator; vi) at least one protein kinase C activator, and vii) a ROCK inhibitor to induce the differentiation of at least some of the primitive gut tube cells into PDXl-positive pancreatic progenitor cells, wherein the PDXl-positive pancreatic progenitor cells express PDX1.

In some aspects, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with one or more of i) at least one BMP signaling pathway inhibitor, ii) a growth factor from TGF-P superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator; and vi) at least one protein kinase C activator, to induce the differentiation of at least some of the primitive gut tube cells into PDX1 -positive pancreatic progenitor cells, wherein the PDXl-positive pancreatic progenitor cells express PDX1.

In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with one or more of i) at least one BMP signaling pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator; and v) at least one protein kinase C activator, to induce the differentiation of at least some of the primitive gut tube cells into PDXl-positive pancreatic progenitor cells, wherein the PDXl-positive pancreatic progenitor cells express PDX1.

In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with i) at least one SHH pathway inhibitor, ii) at least one retinoic acid (RA) signaling pathway activator; and iii) at least one protein kinase C activator, wherein the PDX1 -positive pancreatic progenitor cells express PDX1.

In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with i) at least one growth factor from the FGF family, and ii) at least one retinoic acid (RA) signaling pathway activator, to induce the differentiation of at least some of the primitive gut tube cells into PDXl-positive pancreatic progenitor cells, wherein the PDX1 -positive pancreatic progenitor cells express PDX1.

Any BMP signaling pathway inhibitor capable of inducing primitive gut tube cells to differentiate into PDXl-positive pancreatic progenitor cells (e.g., alone, or with any combination of a growth factor from TGF-P superfamily, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and ROCK inhibitor) can be used in the method provided herein. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor e.g., LDN1931189), such as, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about IpM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., DMH-1), such as, about 0.01 pM, about 0.02pM, about 0.05pM, about 0.1 pM, about 0.2pM, about 0.5 pM, about 0.8 pM, about 1 pM, about 1.2 pM, about 1.5pM, about 1.75pM, about 2 pM, about 2.2 pM, about 2.5pM, about 2.75pM, about 3 pM, about 3.25 pM, about 3.5 pM, about 3.75 pM, about 4 pM, about 4.5 pM, about 5 pM, about 8 pM, about 10 pM, about 15 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., DMH-1), such as, about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., DMH-1) about 250 nM.

Any growth factor from the TGF-P superfamily capable of inducing primitive gut tube cells to differentiate into PDX1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, a growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and ROCK inhibitor) can be used. In some embodiments, the growth factor from TGF-P family comprises Activin A. In some embodiments, the growth factor from TGF-P family comprises GDF8. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A), such as, about 5 ng/mL, about 7.5 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL, about 29 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, or about 100 ng/mL. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from TGF-P superfamily e.g., Activin A), such as, about 17-23 ng/ml, about 18-22 ng/ml, or about 19-21 ng/ml. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A) of about 20 ng/ml.

Any growth factor from the FGF family capable of inducing primitive gut tube cells to differentiate into PDX1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, a growth factor from TGF-P superfamily, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and ROCK inhibitor) can be used. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, about 20-80 ng/ml, about 30-70 ng/ml, about 40-60 ng/ml, or about 45-55 ng/ml. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from FGF family (e.g., KGF) of about 50 ng/ml.

Any SHH pathway inhibitor capable of inducing primitive gut tube cells to differentiate into PDX1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, a growth factor from TGF-P superfamily, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and ROCK inhibitor) can be used. In some embodiments, the SHH pathway inhibitor comprises Santl. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 0.001 pM, about 0.002 pM, about 0.005 pM, about 0.01 pM, about 0.02 pM, about 0.03pM, about 0.05pM, about 0.08 pM, about O. lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.21 pM, about 0.22 pM, about 0.23 pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 220-280 nM, about 230-270 nM, about 240- 260 nM, or about 245-255 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a SHH pathway inhibitor (e.g., Santl) of about 250 nM.

Any RA signaling pathway activator capable of inducing primitive gut tube cells to differentiate into PDX1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one protein kinase C activator, and ROCK inhibitor) can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises contacting primitive gut tube cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 0.02 pM, about 0.1 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.65 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.85 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.4 pM, about 4.6 pM, about 4.8 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 12 pM, about 14 pM, about 15 pM, about 16 pM, about 18 pM, about 20 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid) of about 2 pM. Any PKC activator capable of inducing primitive gut tube cells to differentiate into PDXl-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one RA signaling pathway activator, and ROCK inhibitor) can be used. In some embodiments, the PKC activator comprises PdBU. In some embodiments, the PKC activator comprises TPPB. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB), such as, about 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 pM, 10 pM, about 20 pM, about 50 pM, about 75 pM, about 80 pM, about 100 pM, about 120 pM, about 140 pM, about 150 pM, about 175 pM, about 180 pM, about 200 pM, about 210 pM, about 220 pM, about 240 pM, about 250 pM, about 260 pM, about 280 pM, about 300 pM, about 320 pM, about 340 pM, about 360 pM, about 380 pM, about 400 pM, about 420 pM, about 440 pM, about 460 pM, about 480 pM, about 500 pM, about 520 pM, about 540 pM, about 560 pM, about 580 pM, about 600 pM, about 620 pM, about 640 pM, about 660 pM, about 680 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In some embodiments, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB) of 10 nM-1 mM, 10 nM-500 pM, 10 nM-1 pM, 10-800 nM, 100-900 nM, 300-800 nM, 300-600 nM, 400-600 nM, 450-550 nM, or about 500 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB), such as, about 450-550 mM, about 475-525 nM, about 490-510 nM, or about 495-505 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB) of about 500 nM. In some embodiments, primitive gut tube cells are not treated with a PKC activator (e.g., PDBU).

Any ROCK inhibitor capable of inducing primitive gut tube cells to differentiate into PDXl-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, PKC activator, and at least one RA signaling pathway activator) can be used. In some embodiments, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil/HA1077, or H-l 152. In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 26 pM, about 27 pM, about 28 pM, about 29 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 pM, about 2.3- 2.7 pM, or about 2.4-2.6 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a ROCK inhibitor e.g., Y-27632 or Thiazovivin) of about 2.5 pM.

In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some cases, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA Is 80% hydrolyzed.

In some embodiments, PDX1 -positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDX1 -positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with retinoic acid, KGF, Santl, DMH-1, PdBU, thiazovivin, and Activin A, for a suitable period of time, e.g., about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, PDX1- positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDX1 -positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with retinoic acid, KGF, Santl, DMH-1, PdBU, thiazovivin, and Activin A, for about 2 days. In some embodiments, PDX1 -positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with retinoic acid, KGF, Santl, DMH-1, PdBU, thiazovivin, and Activin A for 1 day, followed by contacting the cells with retinoic acid, KGF, Santl, PdBU, thiazovivin, and Activin A for 1 day (in the absence of DMH-1).

NKX6.1 -positive Pancreatic Progenitor Cells

Aspects of the disclosure involve NKX6.1 -positive pancreatic progenitor cells. NKX6.1 -positive pancreatic progenitor cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, PDX1 -positive, NKX6.1 -negative pancreatic progenitor cells are differentiated to PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells. In some aspects, the NKX6.1 -positive pancreatic progenitor cells are further differentiated, e.g., to Ngn3-positive endocrine progenitor cells, or insulin-positive endocrine cells, followed by induction or maturation to SC-P cells.

In some aspects, a method of producing a NKX6.1 -positive pancreatic progenitor cell from a PDX1 -positive pancreatic progenitor cell comprises contacting a population of cells e.g., under conditions that promote cell clustering and/or promoting cell survival) comprising PDX1 -positive pancreatic progenitor cells with at least two P celldifferentiation factors comprising a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic acid (RA) signaling pathway activator, to induce the differentiation of at least one PDX1 -positive pancreatic progenitor cell in the population into NKX6.1 -positive pancreatic progenitor cells, wherein the NKX6.1 -positive pancreatic progenitor cells expresses NKX6.1.

In some embodiments, the PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDX1 -positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, to induce the differentiation of at least some of the PDXl-positive pancreatic progenitor cells into PDX1 -positive, NKX6.1- positive pancreatic progenitor cells, wherein the PDXl-positive, NKX6.1- positive pancreatic progenitor cells express PDX1 and NKX6.1.

In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-P superfamily, to induce the differentiation of at least some of the PDX1 -positive pancreatic progenitor cells into PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells. In some embodiments, following 3, 4, or 5 days of contacting the PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDX1 -positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-P superfamily; the cells are then contacted with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-P superfamily, and vi) a PKC activator and optionally vii) a gamma-secretase inhibitor. In some embodiments, the PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDX1 -positive pancreatic progenitor cells under conditions that promote cell clustering with at least one growth factor from the FGF family. In some embodiments, the growth factor from the FGF family is KGF.

In some embodiments, the disclosure provides for a method in which a first population of cells comprising PDX1 -positive, NKX6.1 -negative cells is cultured in a media comprising any one or combination of: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) a RA signaling pathway activator, iv) a ROCK inhibitor, and v) a growth factor from the TGF-P superfamily for a period of about 1, 2, 3, 4 or 5 days (e.g., 2-4, 3-4, or 4-5 days); thereby generating a second population of cells. In some embodiments, the second population of cells is then incubated in a composition comprising any one or combination of: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) a RA signaling pathway activator, iv) a ROCK inhibitor, v) a growth factor from the TGF-P superfamily, vi) a PKC activator, vii) a FoxOl inhibitor, and optionally viii) a notch signaling inhibitor for about 1, 2, or 3 days (e.g., 1-2, 1-3, or 2-3 days).

In some embodiments, in the media for culturing the first population of cells, the growth factor from the FGF family is present at a concentration of about 45-55 ng/ml, about 46-54 ng/ml, about 47-53 ng/ml, about 48-52 ng/ml, or about 49-51 ng/ml, the SHH pathway inhibitor is present at a concentration of about 200-300 nM, about 220-280 nM, or about 240-260 nM, the RA signaling pathway activator is present at a concentration of about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM, the ROCK inhibitor is present at a concentration of about 2-3 pM, about 2.2-2.8 pM, or about 2.4-2.6 pM, and/or the growth factor from the TGF-P superfamily is present at a concentration of about 2-8 ng/ml, about 3-7 ng/ml or about 4-6 ng/ml.

In some embodiments, in the media for culturing the second population of cells, the growth factor from the FGF family is present at a concentration of about 45-55 ng/ml, about 46-54 ng/ml, about 47-53 ng/ml, about 48-52 ng/ml, or about 49-51 ng/ml, the SHH pathway inhibitor is present at a concentration of about 200-300 nM, about 220-280 nM, or about 240-260 nM, the RA signaling pathway activator is present at a concentration of about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM, the ROCK inhibitor is present at a concentration of about 2-3 pM, about 2.2-2.8 pM, or about 2.4-2.6 pM, the growth factor from the TGF-P superfamily is present at a concentration of 2 about -8 ng/ml, about 3-7 ng/ml or about 4-6 ng/ml, the PKC activator is present at a concentration of about 0.2- 0.8 pM, about 0.3-0.7 pM, or about 0.4-0.6 pM, and the FoxOl inhibitor is present at a concentration of about 0.7-1.3 pM, about 0.8-1.2 pM, or about 0.9-1.1 pM, and optionally the notch signaling inhibitor is present at a concentration of about 1.7-2.3 pM, about 1.8- 2.2 pM, or about 1.9-2.1 pM.

In some embodiments, the PDX1 -positive pancreatic progenitor cells are produced from a population of pluripotent cells. In some embodiments, the PDX1 -positive pancreatic progenitor cells are produced from a population of iPS cells. In some embodiments, the PDX1 -positive pancreatic progenitor cells are produced from a population of ESC cells. In some embodiments, the PDX1 -positive pancreatic progenitor cells are produced from a population of definitive endoderm cells. In some embodiments, the PDX1 -positive pancreatic progenitor cells are produced from a population of primitive gut tube cells.

Any growth factor from the FGF family capable of inducing PDX1 -positive pancreatic progenitor cells to differentiate into NKX6.1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one SHH pathway inhibitor, a ROCK inhibitor, a growth factor from the TGF-P superfamily, and at least one retinoic acid signaling pathway activator) can be used in the method provided herein. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF8B, FGF 10, and FGF21. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a growth factor from FGF family e.g., KGF), such as, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, about 20-80 ng/ml, about 30-70 ng/ml, about 40-60 ng/ml, or about 45-55 ng/ml. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a growth factor from FGF family (e.g., KGF) of about 50 ng/ml.

Any SHH pathway inhibitor capable of inducing PDX1 -positive pancreatic progenitor cells to differentiate into NKX6.1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, a retinoic acid signaling pathway activator, ROCK inhibitor, and at least one growth factor from the TGF-P superfamily) can be used in the method provided herein. In some embodiments, the SHH pathway inhibitor comprises Santl. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 0.001 pM, about 0.002 pM, about 0.005 pM, about 0.01 pM, about 0.02 pM, about 0.03pM, about 0.05pM, about 0.08 pM, about O. lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about

0.21 pM, about 0.22pM, about 0.23 pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about

0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl) of about 250 nM.

Any RA signaling pathway activator capable of inducing PDXl-positive pancreatic progenitor cells to differentiate into NKX6.1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one SHH pathway inhibitor, ROCK inhibitor, and at least one growth factor from the TGF-P superfamily) can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 0.02 pM, about 0.1 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.65 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.85 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.4 pM, about 4.6 pM, about 4.8 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 12 pM, about 14 pM, about 15 pM, about 16 pM, about 18 pM, about 20 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 70-130 nM, about 80-120 nM, about 90-110 nM, or about 95-105 nM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid) of about 100 nM.

Any ROCK inhibitor capable of inducing PDX1 -positive pancreatic progenitor cells to differentiate into NKX6.1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a RA signaling pathway activator, and at least one growth factor from the TGF-P superfamily) can be used. In some embodiments, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil/HA1077, or 14-1152. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 26 pM, about 27 pM, about 28 pM, about 29 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 pM, about 2.3-2.7 pM, or about 2.4-2.6 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) of about 2.5 pM.

Any activator from the TGF-P superfamily capable of inducing PDXl-positive pancreatic progenitor cells to differentiate into NKX6.1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a RA signaling pathway activator, and ROCK inhibitor) can be used. In some embodiments, the activator from the TGF-P superfamily comprises Activin A or GDF8. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A), such as, about 0.1 ng/mL, about 0.2 ng/mL, about 0.3 ng/mL, about 0.4 ng/mL, about 0.5 ng/mL, about 0.6 ng/mL, about 0.7 ng/mL, about 0.8 ng/mL, about 1 ng/mL, about 1.2 ng/mL, about 1.4 ng/mL, about 1.6 ng/mL, about 1.8 ng/mL, about 2 ng/mL, about 2.2 ng/mL, about 2.4 ng/mL, about 2.6 ng/mL, about 2.8 ng/mL, about 3 ng/mL, about 3.2 ng/mL, about 3.4 ng/mL, about 3.6 ng/mL, about 3.8 ng/mL, about 4 ng/mL, about 4.2 ng/mL, about 4.4 ng/mL, about 4.6 ng/mL, about 4.8 ng/mL, about 5 ng/mL, about 5.2 ng/mL, about 5.4 ng/mL, about 5.6 ng/mL, about 5.8 ng/mL, about 6 ng/mL, about 6.2 ng/mL, about 6.4 ng/mL, about 6.6 ng/mL, about 6.8 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, or about 50 ng/mL. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A), such as, about 2-8 ng/ml, about 3-7 ng/ml, about 4-6 ng/ml, or about 4.5-5.5 ng/ml. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A), such as, about 5 ng/mL.

Any FoxOl inhibitor capable of inducing PDXl-positive pancreatic progenitor cells to differentiate into NKX6.1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, at least one growth factor from the TGF-P superfamily, PKC activator, and Notch signaling inhibitor) can be used in the method provided herein. In some embodiments, the FoxOl inhibitor is AS1842856. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a FoxOl inhibitor (e.g., AS1842856), such as, about O.lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.21 pM, about 0.22pM, about 0.23 pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a FoxOl inhibitor (e.g., AS1842856), such as, about 0.7-1.3 pM, about 0.8-1.2 pM, about or 0.9-1.1 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a FoxOl inhibitor (e.g., AS1842856), such as, about 1 pM.

Any PKC activator capable of inducing PDXl-positive pancreatic progenitor cells to differentiate into NKX6.1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, at least one growth factor from the TGF-P superfamily, FoxOl inhibitor, and Notch signaling inhibitor) can be used in the method provided herein. In some embodiments, the PKC activator is PDBU. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a PKC activator (e.g., PDBU), such as, about 0.1 pM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.2 IpM, about 0.22pM, about 0.23 pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a PKC activator (e.g., PDBU), such as, about 0.2-0.8 pM, about 0.3-0.7 pM, about 0.4-0.6 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a PKC activator (e.g., PDBU), such as, about 0.5 pM. Any Notch signaling inhibitor capable of inducing PDXl-positive pancreatic progenitor cells to differentiate into NKX6.1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, at least one growth factor from the TGF-P superfamily, FoxOl inhibitor, and PKC activator) can be used in the method provided herein. In some embodiments, the Notch signaling inhibitor is XXI. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a Notch signaling inhibitor (e.g., XXI), such as, about 0.1 pM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.21 pM, about 0.22 pM, about 0.23 pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a Notch signaling inhibitor (e.g., XXI), such as, about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9- 2.1 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a Notch signaling inhibitor (e.g., XXI), such as, about 2 pM.

In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells under conditions that promote cell clustering with KGF, Santl, and RA, for a period of 5 days or 6 days. In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells under conditions that promote cell clustering with KGF, Santl, RA, thiazovivin, and Activin A, for a period of 5 or 6 days. In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells under conditions that promote cell clustering with KGF for a period of 5 days. In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells under conditions that promote cell clustering with KGF for a period of 6 days. In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by: a) contacting PDXl-positive pancreatic progenitor cells with KGF, Santl, RA, thiazovivin, and Activin A, for a period of 3, 4 or 5 days (e.g., 4 days), followed by; b) contacting the cells of a) with PDBU, XXI, KGF, Santl, RA, thiazovivin, and Activin A and optionally AS 1842856 for a period of 1, 2 or 3 days (e.g., 2 days).

Insulin-positive Endocrine Cells

Aspects of the disclosure involve insulin-positive endocrine cells (e.g., NKX6.1- positive, ISL1 -positive cells, or P-like cells) and additional methods of generating insulinpositive endocrine cells. Insulin-positive endocrine cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some aspects, NKX6.1 -positive pancreatic progenitor cells are differentiated to insulin-positive endocrine cells (e.g., NKX6.1 -positive, ISLl-positive cells, or P-like cells), In some aspects, the insulin-positive endocrine cells are further differentiated, e.g., by induction or maturation to SC-P cells.

In some aspects, a method of producing an insulin-positive endocrine cell from an NKX6.1 -positive pancreatic progenitor cell comprises contacting a population of cells (e.g., under conditions that promote cell clustering) comprising NKX6-l-positive pancreatic progenitor cells with a) a TGF-P signaling pathway inhibitor, b) a thyroid hormone signaling pathway activator, , c) a BMP pathway inhibitor, and/or d) a protein kinase inhibitor to induce the differentiation of at least one NKX6.1 -positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine ceil expresses insulin. In some embodiments, insulin-positive endocrine cells express PDX1, NKX6.1, ISL1, NKX2.2, Mafb, glis3, Suri, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

Any TGF-P signaling pathway inhibitor capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with other P cell-differentiation factors, e.g., a thyroid hormone signaling pathway activator) can be used. In some embodiments, the TGF-P signaling pathway comprises TGF-P receptor type I kinase signaling. In some embodiments, the TGF-P signaling pathway inhibitor comprises Alk5 inhibitor II. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 0.1 pM, about 0.5 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 10.5 pM, about 11 pM, about 11.5 pM, about 12 pM, about 12.5 pM, about 13 pM, about 13.5 pM, about 14 pM, about 14.5 pM, about 15 pM, about 15.5 pM, about 16 pM, about 16.5 pM, about 17 pM, about 17.5 pM, about 18 pM, about 18.5pM, about 19 pM, about 19.5 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, or about 50 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 7-13 pM, about 8-12 pM, about 9-11 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 10 pM.

Any thyroid hormone signaling pathway activator capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells to differentiate into insulinpositive endocrine cells (e.g., alone, or in combination with other P cell-differentiation factors, e.g., a TGF-P signaling pathway inhibitor) can be used. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator comprises GC-1. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about O. lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.21 pM, about 0.22pM, about 0.23 pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 0.7-1.3 pM, about 0.8-1.2 pM, or about 0.9-1.1 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 1 pM.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive pancreatic progenitor cells) with at least one additional factor. In some embodiments, the method comprises contacting the PDX1 -positive NKX6.1 -positive pancreatic progenitor cells with at least one of i) a SHH pathway inhibitor, ii) a y-secretase inhibitor, iii) at least one growth factor from the epidermal growth factor (EGF) family, iv) a TGF-P signaling pathway inhibitor, or vii) a thyroid hormone signaling pathway activator. In some embodiments, the method comprises contacting the population of cells e.g., NKX6.1 -positive pancreatic progenitor cells) with at least one additional factor. In some embodiments, the method comprises contacting the PDX1 -positive NKX6.1 -positive pancreatic progenitor cells with at least one of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a y-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a TGF-P signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) a wnt signaling pathway inhibitor, or ix) a PKC activator.

In some embodiments, the method comprises contacting the PDX1 -positive NKX6.1 -positive pancreatic progenitor cells with at least one of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a y-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-P signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) a protein kinase inhibitor, or ix) a ROCK inhibitor.

In some embodiments, the method comprises contacting the PDX1 -positive NKX6.1 -positive pancreatic progenitor cells with at least one of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a y-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-P signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, or x) a ROCK inhibitor. In some embodiments, the method comprises contacting the PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells in a culture with a i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a y-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-P signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, x) a ROCK inhibitor, xi) a PKC activator and xii) a Wnt signaling pathway inhibitor for 1, 2, or 3 days (e.g., 1-2, 1-3, or 2-3 days), and then contacting the cells in the culture with i) a y-secretase inhibitor, ii) at least one growth factor from the epidermal growth factor (EGF) family, iii) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, iv) a TGF-P signaling pathway inhibitor, v) a thyroid hormone signaling pathway activator, vi) an epigenetic modifying compound, vii) a protein kinase inhibitor, and viii) a ROCK inhibitor for a period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 1-7, 1- 5, 1-3, 3-7, 3-5, 5-7, or 4-6 days) in the absence of a SHH pathway inhibitor, a RA signaling pathway activator, a Wnt signaling pathway inhibitor, PKC activator, and/or growth factor from the epidermal growth factor (EGF) family.

In some embodiments, in the method of generating the insulin-positive endocrine cells from the PDXl-positive NKX6.1-postive pancreatic progenitor cells, some of the differentiation factors are present only for the first 1, 2, 3, 4, or 5 days during the differentiation step. In some embodiments, some of the differentiation factors, such as the SHH pathway inhibitor, the RA signaling pathway activator, the PKC activator, and the at least one growth factor from the EGF family are removed from the culture medium after the first 1, 2, or 3 days of incubation.

Any y-secretase inhibitor that is capable of inducing the differentiation of NKX6.1- positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the y- secretase inhibitor comprises XXI. In some embodiments, the y-secretase inhibitor comprises DAPT. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a y-secretase inhibitor (e.g., XXI), such as, about 0.01 pM, about 0.02 pM, about 0.05 pM, about 0.075 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.4 pM, about 4.6 pM, about 4.8 pM, about 5 pM, about 5.2 pM, about 5.4 pM, about 5.6 pM, about 5.8 pM, about 6 pM, about 6.2 pM, about 6.4 pM, about 6.6 pM, about 6.8 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 20 pM, about 30 pM, or about 50 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a y-secretase inhibitor (e.g., XXI), such as, about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a y- secretase inhibitor (e.g., XXI), such as about 2 pM.

Any growth factor from the EGF family capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the at least one growth factor from the EGF family comprises betacellulin. In some embodiments, at least one growth factor from the EGF family comprises EGF. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a growth factor from EGF family (e.g., betacellulin), such as, about 1 ng/mL, about 2 ng/mL, about 4 ng/mL, about 6 ng/mL, about 8 ng/mL, about 10 ng/mL, about 12 ng/mL, about 14 ng/mL, about 16 ng/mL, about 18 ng/mL, about 20 ng/mL, about 22 ng/mL, about 24 ng/mL, about 26 ng/mL, about 28 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a growth factor from EGF family (e.g., betacellulin), such as, about 17-23 ng/ml, about 18-22 ng/ml, or about 19-21 ng/ml. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a growth factor from EGF family (e.g., betacellulin), such as, about 20 ng/ml.

Any RA signaling pathway activator capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the RA signaling pathway activator comprises RA. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 0.02 pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.65 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.85 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.4 pM, about 4.6 pM, about 4.8 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 12 pM, about 14 pM, about 15 pM, about 16 pM, about 18 pM, about 20 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 20-80 nM, about 30-70 nM, or about 40-60 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 50 nM.

Any SHH pathway inhibitor capable of inducing the differentiation of NKX6.1- positive pancreatic progenitor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used in the method provided herein. In some embodiments, the SHH pathway inhibitor comprises Santl. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 0.001 pM, about 0.002 pM, about 0.005 pM, about 0.01 pM, about 0.02 pM, about 0.03pM, about 0.05pM, about 0.08 pM, about O. lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.21 pM, about 0.22pM, about 0.23 pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 250 nM.

Any BMP signaling pathway inhibitor capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the BMP signaling pathway inhibitor comprises LDN 193189 or DMH- 1. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of BMP signaling pathway inhibitor e.g., LDN1931189), such as, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about IpM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 70- 130 nM, about 80-120 nM, about 90-110 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 100 nM.

Any ROCK inhibitor that is capable of inducing the differentiation of NKX6.1- positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil/HA1077, or H-l 152. In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 26 pM, about 27 pM, about 28 pM, about 29 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 pM, about 2.3-2.7 pM, or about 2.4-2.6 pM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting PDX1- positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.5 pM.

Any epigenetic modifying compound that is capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor or a HD AC inhibitor. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor, e.g., DZNep. In some embodiments, the epigenetic modifying compound comprises a HD AC inhibitor, e.g., KD5170. In some examples, the method comprises contacting PDX1 -positive, NKX6.1- positive pancreatic progenitor cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 0.01 pM, about 0.025 pM, about 0.05 pM, about 0.075 pM, about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1- positive pancreatic progenitor cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 70-130 nM, about 80-120 nM, or about 90-110 nM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 100 nM.

Any Wnt signaling pathway inhibitor that is capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the Wnt signaling pathway inhibitor comprises a tankyrase inhibitor. In some embodiments, the tankyrase inhibitor is NVP-TNKS656. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), such as, about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.65 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.85 pM, about 0.9 pM, about 0.95 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, or about 5 pM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), such as, about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), such as, about 2 pM.

Any PKC activator that is capable of inducing the differentiation of NKX6.1- positive pancreatic progenitor cells in a population into insulin-positive endocrine cells e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the PKC activator is TPB or PDBU. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a PKC activator (TPB or PDBU), such as, about 0.01 pM, about 0.025 pM, about 0.05 pM, about 0.075 pM, about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.65 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.85 pM, about 0.9 pM, about 0.95 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, or about 20 pM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a PKC activator (TPB or PDBU), such as, about 450-550 mM, about 475-525 nM, about 490-510 nM, or about 495-505 nM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a PKC activator (TPB or PDBU), such as, about 500 nM.

In some embodiments, the population of cells is optionally contacted with a protein kinase inhibitor. In some embodiments, the population of cells is not contacted with the protein kinase inhibitor. In some embodiments, the population of cells is contacted with the protein kinase inhibitor. Any protein kinase inhibitor that is capable of inducing the differentiation of NKX6. 1 -positive pancreatic progenitor cells in a population into insulinpositive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator). In some embodiments, the protein kinase inhibitor comprises staurosporine. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0 nM, about 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM, about 2.8 pM, about 2.9 nM, about 3 nM, about 3.1 nM, about 3.2 nM, about 3.3 nM, about 3.4 nM, about 3.5 nM, about 3.6 nM, about 3.7 nM, about 3.8 nM, about 3.9 nM, about 4.0 nM, about 4.1 nM, about 4.2 nM, about 4.3 nM, about 4.4 nM, about 4.5 nM, about 4.6 nM, about 4.7 nM, about 4.8 pM, about 4.9 nM, or about 5 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 1-5 nM, about 2-4 nM, or about 2.5-3.5 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 3 nM.

In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some cases, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 89% hydrolyzed.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Santl, and betacellulin, PDBU, and NVP-TNKS656 for a period of 7 days, to induce the differentiation of at least one NKX6.1 -positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Santl, betacellulin, and LDN193189 for a period of 7 days, to induce the differentiation of at least one NKX6.1 -positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, one or more differentiation factors are added in a portion of the Stage 5, for instance, only the first 1, 2, 3, 4, 5, or 6 days of the period of time for Stage 5, or the last 1, 2, 3, 4, 5, or 6 days of the period of time for Stage 5. In one example, the cells are contacted with SHH signaling pathway inhibitor the PKC activator, the retinoic acid, and/or the wnt signaling pathway inhibitor for only the first 2, 3, 4, or 5 days during Stage 5, after which the SHH signaling pathway inhibitor, the PKC activator, the retinoic acid, and/or the wnt signaling pathway inhibitor are not included in or removed from the culture medium. In another example, the cells are contacted with BMP signaling pathway inhibitor for only the first 1, 2, or 3 days during Stage 5, after which the BMP signaling pathway inhibitor is removed from the culture medium.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive pancreatic progenitor cells) with one or more metabolites. In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1- positive pancreatic progenitor cells) with one or more of an acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, and/or glutamine. Examples of metabolites include glutamine, taurine, acetate, beta-hydroxybutyrate, biotin, and formate. In some embodiments, a composition (e.g., medium) of the disclosure comprises an acetyl CoA-related metabolite. Exemplary acetyl CoA-related metabolites include, but are not limited to acetate, pyruvate, ketogenic amino acids, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine, tryptophan, fatty acids, CoA, Isovaleryl-CoA, and P- hydroxybutyrate. In some embodiments, the acetyl CoA-related metabolite is acetate. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 10 nM, about 50 nM, about 80 nM, about 100 nM, about 120 nM, about 140 nM, about 150 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1 pM, about 10 pM, about 100 pM, about 500 pM, about 800 pM, about 900 pM, about 1 mM, about 2 mM, about 3 mM, about 5 mM, or about 10 mM. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9- 1.1 mM, or 0.95-1.05 mM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 1 mM. In some embodiments, the acetyl CoA- related metabolite is acetate present at a concentration of about 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 160 nM.

In some embodiments, a composition (e.g., medium) of the disclosure comprises one or more vitamins. Exemplary vitamins include, but are not limited to biotin, vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine) and vitamin B 12 (cyanocobalamin). In some embodiments the vitamin modulates fatty acid synthesis. In some embodiments the vitamin modulates branched-chain amino acid metabolism. In some embodiments the vitamin modulates or participates as a co-factor in the TCA cycle, e.g., as a cofactor for pyruvate carboxylase. In some embodiments, the vitamin is biotin. In some embodiments, the vitamin is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 1.5 pM, about 3 pM, about 5 pM, about 10 pM, or about 100 pM. In some embodiments, the vitamin is biotin present at a concentration of about 800 nM. In some embodiments, the vitamin is present in or is added to a composition of the disclosure at a concentration of about 1 nM to 500 pM, 1 nM to 100 pM, 1 nM to 10 pM, 1 nM to 1 pM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 pM, 25 nM to 100 pM, 25 nM to 10 pM, 25 nM to 1 pM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 pM, 50 nM to 100 pM, 50 nM to 10 pM, 50 nM to 1 pM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 pM, 100 nM to 100 pM, 100 nM to 10 pM, 100 nM to 1 pM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

In some embodiments, a composition (e.g., medium) of the disclosure comprises a histone deacetylase inhibitor (HDACi). Exemplary histone deacetylase inhibitors (HDACi) include, but are not limited to P-Hydroxybutyrate, butyric acid, class I HDACi, class IIA HDACi, class IIB HDACi, class III HDACi, class IV HDACi, HDAC-1, HDAC- 2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10, HDAC-11, sirtuins, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, Vorinostat (suberoylanilide hydroxamic acid, SAHA, MK0683), Entinostat (MS-275, SNDX-275), Panobinostat (LBH589, NVP-LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391 A, HDAC-IN-3), BRD3308, BRD3308, Tubastatin A TFA (Tubastatin A trifluoroacetate salt), Tubastatin A, SIS 17, NKL 22, BML-210 (CAY10433), TC-H 106, SR-4370, Belinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR 901228, NSC 630176), MC1568, Givinostat (ITF2357), Dacinostat (LAQ824, NVP-LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), PCI-34051, Droxinostat (NS 41080), Abexinostat (PCI- 24781), Abexinostat (PCI-24781, CRA-024781), RGFP966, AR-42 (HD AC-42), Ricolinostat (ACY-1215, Rocilinostat), Valproic acid sodium salt (Sodium valproate), Tacedinaline (CI994, PD-123654, GOE-5549, Acetyldinaline), Fimepinostat (CUDC- 907), Sodium butyrate (NaB), Curcumin, Diferuloylmethane, M344, Tubacin, RG2833 (RGFP109), RG2833 (RGFP109), Resminostat (RAS2410), Divalproex Sodium, Scriptaid (GCK 1026), Sodium Phenylbutyrate, Sinapinic acid (Sinapic acid), TMP269, Santacruzamate A (CAY10683), TMP195 (TFMO 2), Valproic acid (VP A), UF010, Tasquinimod (ABR-215050), SKLB-23bb, Isoguanosine, Sulforaphane, BRD73954, Citarinostat (ACY-241, HDAC-IN-2), Suberohydroxamic acid, Splitomicin, HPOB, LMK-235, Biphenyl-4-sulfonyl chloride (p-Phenylbenzenesulfonyl, 4- Phenylbenzenesulfonyl, p-Biphenyl sulfonyl), Nexturastat A, TH34, Tucidinostat (Chidamide, HBI-8000, CS-055), (-)-Parthenolide, WT161, CAY10603, CAY10603, ACY-738, Raddeanin A, Tinostamustine(EDO-S101), Domatinostat (4SC-202), and BG45. In some embodiments, the HDACi is P-Hydroxybutyrate. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 1.5 pM, about 3 pM, about 5 pM, about 10 pM, or about 100 pM. In some embodiments, the HDACi is P-Hydroxybutyrate present at a concentration of about 200 nM. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 1 nM to 500 pM, 1 nM to 100 pM, 1 nM to 10 pM, 1 nM to 1 pM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 5 nM to 500 pM, 25 nM to 100 pM, 25 nM to 10 pM, 25 nM to 1 pM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 pM, 50 nM to 100 pM, 50 nM to 10 pM, 50 nM to 1 pM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 pM, 100 nM to 100 pM, 100 nM to 10 pM, 100 nM to 1 pM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

In some embodiments, a composition (e.g., medium) of the disclosure comprises a redox homeostasis regulator. Exemplary redox homeostasis regulators include, but are not limited to taurine, respiratory chain regulators, free radical scavengers, regulators of mitochondrial protein synthesis, allium sulphur compounds, anthocyanins, beta-carotene, catechins, copper, cryptoxanthins, flavonoids, indoles, isoflavonoids, lignans, lutein, lycopene, alpha lipoic acid, ellagic acid, manganese, polyphenols, selenium, glutathione, vitamin A, vitamin C, vitamin E, zinc, superoxide disutases, GSHPx, Prx-I, catalase, and co-enzyme Q10. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 500 nM, 1 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 110 pM, about 150 pM, or about 200 pM. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is taurine present at a concentration of about 90 pM. In some embodiments, the redox homeostasis regulator intermediate is present or is added at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM, 1 pM to 1 mM, 10 pM to 1 mM, 20 pM to 1 mM, 30 pM to 1 mM, 30 pM to 1 mM, 40 pM to 1 mM, 50 pM to 1 mM, 60 pM to 1 mM, 70 pM to 1 mM, 80 pM to 1 mM, 100 nM to 250 pM, 500 nM to 250 pM, 1 pM to 250 pM, 10 pM to 250 pM, 20 pM to 250 pM, 30 pM to 250 pM, 30 pM to 250 pM, 40 pM to 250 pM, 50 pM to 250 pM, 60 pM to 250 pM, 70 pM to 250 pM, 100 nM to 100 pM, 500 nM to 100 pM, 1 pM to 100 pM, 10 pM to 100 pM, 20 pM to 100 pM, 30 pM to 100 pM, 40 pM to 100 pM, 50 pM to 100 pM, 60 pM to 100 pM, 70 pM to 100 pM, or 80 pM to 100 pM.

In some embodiments, a composition (e.g., medium) of the disclosure comprises a one carbon metabolism pathway intermediate. Exemplary one carbon metabolism pathway intermediates include, but are not limited to formate, tetrahydrofolate (THF), 10- formylTHF; 5,10-meTHF; 5,10-meTHF; and 10-formylTHF. In some embodiments, the one carbon metabolism pathway intermediate is formate present at a concentration of about 50 pM. In some embodiments, the one carbon metabolism pathway intermediate is present or is added at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM, 1 pM to 1 mM, 10 pM to 1 mM, 20 pM to 1 mM, 30 pM to 1 mM, 100 nM to 250 pM, 500 nM to 250 pM, 1 pM to 250 pM, 10 pM to 250 pM, 20 pM to 250 pM, 30 pM to 250 pM, 100 nM to 100 pM, 500 nM to 100 pM, 1 pM to 100 pM, 10 pM to 100 pM, 20 pM to 100 pM, 30 pM to 100 pM, 100 nM to 60 pM, 500 nM to 60 pM, 1 pM to 60 pM, 10 pM to 60 pM, 20 pM to 60 pM, 30 pM to 60 pM, 40 pM to 60 pM, or 45 pM to 55 pM.

In some embodiments, a composition (e.g., medium) of the disclosure comprises glutamine. Thus in some embodiments, compositions and methods of the disclosure utilize glutamine in a form with increased bioavailability, such as a free glutamine form, such as a non-dipeptide form, a non-alanine-glutamine dipeptide form (e.g., a non-alanyl-

1-glutamine form), a non-glycine-glutamine dipeptide form (e.g., a non-glycyl-l-glutamine form), a form that in which glutamine is not conjugated to another amino acid or stabilizing moiety, a monomeric form, a free form, or a combination thereof. In some embodiments, glutamine is provided as a protein hydrolysate. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of from 0.5-20 mM, 0.5-10 mM, 0.5-5 mM, 1-5 mM, 2-5 mM, or 1 mM to 10 mM. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of 3.8-4.2 mM. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of 1-10, 1-7, 1-8, 1-6, 1-5, 1-4, 2-10, 2-7,

2-8, 2-6, 2-5, 2-4, 3-10, 3-7, 3-8, 3-6, 3-5, 3-4, 3.5-4.5, 3.8-4.2, or 3.9-4.1 mM. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of about 4 mM. In some embodiments, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in a dipeptide form. In some embodiments, at least 500 pM, at least 750 pM, at least 1 mM, at least 1.5 mM, at least 2 mM, at least 2.5 mM, at least 2.6 mM, at least 2.7 mM, at least 2.8 mM, at least 2.9 mM, at least 3 mM, at least 3.1 mM, at least 3.2 mM, at least 3.3 mM, at least 3.4 mM, at least 3.5 mM, at least 3.6 mM, at least 3.7 mM, at least 3.8 mM, at least 3.9 mM, at least 4 mM, at least 5 mM, at least 5.5 mM, at least 6 mM, at least 6.5 mM, at least 7 mM, at least 7.5 mM, at least 8 mM, at least 8.5 mM, at least 9 mM, at least 9.5 mM, or at least 10 mM of the glutamine is in a free form.

In some embodiments, the method comprises culturing the population of cells (e.g., NKX6.1 -positive pancreatic progenitor cells) in a medium, to induce the differentiation of at least one NKX6.1 -positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin.

Aspects of the disclosure involve treatment of cell population comprising PDX1- positive, NKX6.1 -positive pancreatic progenitor cells with PKC activator and/or wnt signaling pathway inhibitor, which can lead to increase in percentage of pancreatic a cells, increase in percentage of pancreatic 5 cells, increase in percentage of pancreatic P cells, reduction in percentage of EC cells, or any combination thereof, in the cell population of pancreatic endocrine cells generated according to the method disclosed herein.

In some embodiments, the method comprises contacting a population of cells comprising PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a first composition comprising a FOXO1 inhibitor, notch signaling inhibitor, a PKC activator, a ROCK inhibitor, a growth factor from TGFP superfamily, a growth factor from FGF family, a RA signaling pathway activator, and a SHH pathway inhibitor, for one to two days, thereby obtaining a first transformation cell population comprising PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells; and contacting the first transformation cell population comprising PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a second composition comprising the PKC activator, notch signaling inhibitor, a TGF-P signaling pathway inhibitor, a TH signaling pathway activator, BMP pathway inhibitor, ROCK inhibitor, retinoic acid, and EGF-family growth factor, wnt signaling pathway inhibitor, and/or an epigenetic modifying compound, for one to two days, thereby obtaining a second transformation cell population comprising NKX6.1 -positive, ISL1- positive endocrine cells.

Pancreatic Cells

Aspects of the disclosure involve generating pancreatic P cells (e.g., non-native pancreatic P cells/SC-P cells) and additional methods of generating them. Non-native pancreatic P cells, In some embodiments, resemble endogenous mature P cells in form and function, but nevertheless are distinct from native P cells.

In some embodiments, the insulin-positive pancreatic endocrine cells generated using the method provided herein can form a cell cluster, alone or together with other types of cells, e.g., precursors thereof, e.g., stem cell, definitive endoderm cells, primitive gut tube cell, PDX1 -positive pancreatic progenitor cells, or NKX6.1 -positive pancreatic progenitor cells.

In some embodiments, any of the cells or populations of cells disclosed herein are in a cell cluster. In some aspects, provided herein are cell clusters that resemble the functions and characteristics of endogenous pancreatic islets. Such cell clusters can mimic the function of endogenous pancreatic islets in regulating metabolism, e.g., glucose metabolism in a subject.

In some embodiments, a composition or cell population of the present disclosure comprises NKX6.1 -positive, ISL-positive cells that express lower levels of MAFA than NKX6.1 -positive, ISL-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the composition or cell population comprises NKX6.1 -positive, ISL-positive cells that express higher levels of MAFB than NKX6.1 -positive, ISL-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the composition or cell population comprises NKX6.1 -positive, ISL-positive cells that express higher levels of SIX2, HOPX, IAPP and/or UCN3 than NKX6.1 -positive, ISL-positive cells from the pancreas of a healthy control adult subject.

In some embodiments, a composition or cell population of the present disclosure comprises NKX6.1 -positive, ISL-positive cells that do not express MAFA. In some embodiments, the composition or cell population comprises NKX6.1 -positive, ISL- positive cells that express MAFB.

In some embodiments, the cell population comprising the insulin-positive endocrine cells can be directly induced to mature into SC-P cells without addition of any exogenous differentiation factors (such as inhibitor of TGF-P signaling pathway, thyroid hormone signaling pathway activator, PKC activator, growth factors from TGF-P superfamily, FGF family, or EGF family, SHH signaling pathway inhibitor, y-secretase inhibitor, ROCK inhibitor, or BMP signaling pathway inhibitor). In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1- positive, ISLl-positive endocrine cells with a serum albumin protein, a TGF-P signaling pathway inhibitor, a SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and/or an epigenetic modifying compound. In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1 -positive, ISLl-positive endocrine cells with human serum albumin protein. In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1 -positive, ISLl-positive endocrine cells with a PKC activator.

In some embodiments, the cell population comprising the insulin-positive endocrine cells can be induced to mature into SC-P cells by contacting the insulin-positive endocrine cells with differentiation factors. The differentiation factors can comprise at least one inhibitor of TGF-P signaling pathway and thyroid hormone signaling pathway activator as described herein. In some embodiments, SC-P cells can be obtained by contacting a population of cells comprising insulin-positive endocrine cells with Alk5i and T3 or GC-1.

In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1 -positive, ISLl-positive endocrine cells with (i) a TGF-P signaling pathway inhibitor, (ii) a thyroid hormone signaling pathway activator, (iii) an epigenetic modifying compound, (iv) a BMP signaling pathway inhibitor, (v) a ROCK inhibitor, and/or (vi) a protein kinase inhibitor (e.g., staurosporine).

In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1 -positive, ISLl-positive endocrine cells with (i) a growth factor from the FGF family, (ii) a TGF-P signaling pathway inhibitor, (iii) a thyroid hormone signaling pathway activator, (iv) an epigenetic modifying compound, (v) a protein kinase inhibitor, (vi) a ROCK inhibitor, (vii) a BMP signaling pathway inhibitor, and (viii) a lipase inhibitor for about one two five days. In some embodiments, the contacting is for about three days. Any TGF-P signaling pathway inhibitor capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with other P cell-differentiation factors, e.g., a thyroid hormone signaling pathway activator) can be used. In some embodiments, the TGF-P signaling pathway comprises TGF-P receptor type I kinase signaling. In some embodiments, the TGF-P signaling pathway inhibitor comprises Alk5 inhibitor II. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 0.1 pM, about 0.5 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 10.5 pM, about 11 pM, about 11.5 pM, about 12 pM, about 12.5 pM, about 13 pM, about 13.5 pM, about 14 pM, about 14.5 pM, about 15 pM, about 15.5 pM, about 16 pM, about 16.5 pM, about 17 pM, about 17.5 pM, about 18 pM, about 18.5pM, about 19 pM, about 19.5 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, or about 50 pM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 7-13 pM, about 8-12 pM , or about 9-11 pM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 10 pM.

Any thyroid hormone signaling pathway activator capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with other P cell-differentiation factors, e.g., a TGF-P signaling pathway inhibitor) can be used. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator comprises GC-1. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 0.1 pM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.21 pM, about 0.22pM, about 0.23 pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting insulinpositive endocrine cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 0.7-1.3 pM, about 0.8-1.2 pM, or about 0.9-1.1 pM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 1 pM.

Any BMP signaling pathway inhibitor capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about IpM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 70-130 nM, about 80-120 nM, about 90-110 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 100 nM.

Any ROCK inhibitor that is capable of inducing the differentiation of insulinpositive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil/HA1077, or H-l 152. In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 26 pM, about 27 pM, about 28 pM, about 29 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 pM, about 2.3-2.7 pM, or about 2.4-2.6 pM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting insulinpositive endocrine cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.5 pM.

Any epigenetic modifying compound that is capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor or a HD AC inhibitor. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor, e.g., DZNep. In some embodiments, the epigenetic modifying compound comprises a HD AC inhibitor, e.g., KD5170. In some examples, the method comprises contacting insulin-positive endocrine cells to mature into SC-P cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 0.01 M, about 0.025 pM, about 0.05 pM, about 0.075 pM, about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting insulin-positive endocrine cells to mature into SC-P cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 70-130 nM, about 80-120 nM, or about 90-110 nM. In some examples, the method comprises contacting insulin-positive endocrine cells to mature into SC-P cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 100 nM. Any protein kinase inhibitor that is capable of inducing the differentiation insulinpositive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator). In some embodiments, the protein kinase inhibitor comprises staurosporine. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0 nM, about 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM, about 2.8 pM, about 2.9 nM, about 3 nM, about 3.1 nM, about 3.2 nM, about 3.3 nM, about 3.4 nM, about 3.5 nM, about 3.6 nM, about 3.7 nM, about 3.8 nM, about 3.9 nM, about 4.0 nM, about 4.1 nM, about 4.2 nM, about 4.3 nM, about 4.4 nM, about 4.5 nM, about 4.6 nM, about 4.7 nM, about 4.8 pM, about 4.9 nM, or about 5 nM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 1-5 nM, about 2-4 nM, or about 2.5-3.5 nM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 3 nM.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive, ISL1 -positive, insulin-positive cells) with one or more metabolites. In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive, ISL1 -positive, insulin-positive cells) with one or more of an acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, glutamate, and/or carnitine. Examples of metabolites include taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate.

In some embodiments, a composition (e.g., medium) of the disclosure comprises an acetyl CoA-related metabolite. Exemplary acetyl CoA-related metabolites include, but are not limited to acetate, pyruvate, ketogenic amino acids, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine, tryptophan, fatty acids, CoA, Isovaleryl-CoA, and P- hydroxybutyrate. In some embodiments, the acetyl CoA-related metabolite is acetate. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 10 nM, about 50 nM, about 80 nM, about 100 nM, about 120 nM, about 140 nM, about 150 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1 pM, about 10 pM, about 100 pM, about 500 pM, about 800 pM, about 900 pM, about 1 mM, about 2 mM, about 3 mM, about 5 mM, or about 10 mM. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9- 1.1 mM, or 0.95-1.05 mM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 1 mM. In some embodiments, the acetyl CoA- related metabolite is acetate present at a concentration of about 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 160 nM.

In some embodiments, a composition (e.g., medium) of the disclosure comprises a histone deacetylase inhibitor (HDACi). Exemplary histone deacetylase inhibitors (HDACi) include, but are not limited to P-Hydroxybutyrate, butyric acid, class I HDACi, class IIA HDACi, class IIB HDACi, class III HDACi, class IV HDACi, HDAC-1, HDAC- 2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10, HDAC-11, sirtuins, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, Vorinostat (suberoylanilide hydroxamic acid, SAHA, MK0683), Entinostat (MS-275, SNDX-275), Panobinostat (LBH589, NVP-LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391 A, HDAC-IN-3), BRD3308, BRD3308, Tubastatin A TFA (Tubastatin A trifluoroacetate salt), Tubastatin A, SIS 17, NKL 22, BML-210 (CAY10433), TC-H 106, SR-4370, Belinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR 901228, NSC 630176), MC1568, Givinostat (ITF2357), Dacinostat (LAQ824, NVP-LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), PCI-34051, Droxinostat (NS 41080), Abexinostat (PCI- 24781), Abexinostat (PCI-24781, CRA-024781), RGFP966, AR-42 (HD AC-42), Ricolinostat (ACY-1215, Rocilinostat), Valproic acid sodium salt (Sodium valproate), Tacedinaline (CI994, PD-123654, GOE-5549, Acetyldinaline), Fimepinostat (CUDC- 907), Sodium butyrate (NaB), Curcumin, Diferuloylmethane, M344, Tubacin, RG2833 (RGFP109), RG2833 (RGFP109), Resminostat (RAS2410), Divalproex Sodium, Scriptaid (GCK 1026), Sodium Phenylbutyrate, Sinapinic acid (Sinapic acid), TMP269, Santacruzamate A (CAY10683), TMP195 (TFMO 2), Valproic acid (VP A), UF010, Tasquinimod (ABR-215050), SKLB-23bb, Isoguanosine, Sulforaphane, BRD73954, Citarinostat (ACY-241, HDAC-IN-2), Suberohydroxamic acid, Splitomicin, HPOB, LMK-235, Biphenyl-4-sulfonyl chloride (p-Phenylbenzenesulfonyl, 4- Phenylbenzenesulfonyl, p-Biphenyl sulfonyl), Nexturastat A, TH34, Tucidinostat (Chidamide, HBI-8000, CS-055), (-)-Parthenolide, WT161, CAY10603, CAY10603, ACY-738, Raddeanin A, Tinostamustine(EDO-S101), Domatinostat (4SC-202), and BG45. In some embodiments, the HDACi is P-Hydroxybutyrate. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 1.5 pM, about 3 pM, about 5 pM, about 10 pM, or about 100 pM. In some embodiments, the HDACi is P-Hydroxybutyrate present at a concentration of about 200 nM. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 1 nM to 500 pM, 1 nM to 100 pM, 1 nM to 10 pM, 1 nM to 1 pM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 pM, 25 nM to 100 pM, 25 nM to 10 pM, 25 nM to 1 pM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 pM, 50 nM to 100 pM, 50 nM to 10 pM, 50 nM to 1 pM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 pM, 100 nM to 100 pM, 100 nM to 10 pM, 100 nM to 1 pM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

In some embodiments, a composition (e.g., medium) of the disclosure comprises glutamate (e.g., L-glutamate). In some embodiments, glutamate can be present in a composition of the disclosure at a concentration of about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 450 pM, about 500 pM, about 550 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 4 mM, or about 5 mM. In some embodiments, glutamate is present or is added to a composition of the disclosure at a concentration of about 500 pM. In some embodiments, glutamate is present or is added to a composition of the disclosure at a concentration of from about 100 pM to 5mM, 200 pM to 5mM, 300 pM to 5mM, 400 pM to 5mM, 100 pM to 3mM, 200 pM to 3mM, 300 pM to 3mM, 400 pM to 3mM, 100 pM to 2mM, 200 pM to 2mM, 300 pM to 2mM, 400 pM to 2mM, 100 pM to ImM, 200 pM to ImM, 300 pM to ImM, 400 pM to ImM, 100 pM to 700 pM, 200 pM to 700 pM, 300 pM to 700 pM, 400 pM to 700 pM, 100 pM to 600 pM, 200 pM to 600 pM, 300 pM to 600 pM, or 400 pM to 600 pM.

In some embodiments, a composition (e.g., medium) of the disclosure comprises carnitine. In some embodiments, carnitine is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 500 nM, about 1 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 75 pM, or about 100 pM. In some embodiments, carnitine is present or is added at a concentration of about 40 pM. In some embodiments, carnitine is present in or is added to a composition of the disclosure at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM,l pM to 1 mM, 10 pM to 1 mM, 20 pM to 1 mM, 30 pM to 1 mM, 100 nM to 250 pM, 500 nM to 250 pM, 1 pM to 250 pM, 10 pM to 250 pM, 20 pM to 250 pM, 30 pM to 250 pM, 100 nM to 100 pM, 500 nM to 100 pM, 1 pM to 100 pM, 10 pM to 100 pM, 20 pM to 100 pM, 30 pM to 100 pM, 100 nM to 60 pM, 500 nM to 60 pM, 1 pM to 60 pM, 10 pM to 60 pM, 20 pM to 60 pM, 30 pM to 60 pM, 35 pM to 60 pM, or 30 pM to 50 pM.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive, ISL1 -positive, insulin-positive cells) with a serum albumin protein (e.g., HSA). In some embodiments, the serum albumin is present at a concentration of 0.01-2% HSA. In some embodiments, the serum albumin is present at a concentration of 0.03-0.1%, 0.03-0.07%, or 0.04-0.05%. In some embodiments, the serum albumin is present at a concentration of 0.05%. In some embodiments, the serum albumin is present at a concentration of 0.7-1.3%, 0.8-1.2%, 0.9-1.1% or at 1%. In some embodiments, the serum albumin is present at a concentration of 1%.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive, ISL1 -positive, insulin-positive cells) with ZnSC . In some embodiments, the method comprises contacting the cells with 1-100 pM, 1-50 pM, 1-20 pM, 1-12 pM, 5-15 pM, 8-12 pM or 9-11 pM of ZnSCh. In some embodiments, the method comprising contacting the cells with about 10 pM of ZnSCh.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive, ISL1 -positive, insulin-positive cells) with one or more of an a serum albumin protein, a TGF-P signaling pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, an epigenetic modifying compound, acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, glutamate, and/or carnitine for a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some embodiments, the method further comprises contacting the population of cells following the first period with one or more of a serum albumin protein, an acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, glutamate, and/or carnitine for a second period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days) or more in the absence of a TGF-P signaling pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and/or an epigenetic modifying compound. In some embodiments, the cells are contacted with a higher concentration of the serum albumin in the second period as compared to the first period. In some embodiments, the compositions further comprise ZnSO4. In some embodiments, the method further comprises contacting the population of cells following the first period with human serum albumin, but in the absence of a TGF-P signaling pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, an epigenetic modifying compound, an acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, glutamate, and/or carnitine.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive, ISL1 -positive, insulin-positive cells) with one or more of HSA, Alk5 inhibitor II, GC-1, staurosporine, thiazovivin, LDN193189, DZNEP, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate for a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some embodiments, the method further comprises contacting the population of cells following the first period with one or more of HSA, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate for a second period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days) or more in the absence of an Alk5 inhibitor II, GC-1, staurosporine, thiazovivin, LDN193189, DZNEP. In some embodiments, the compositions further comprise ZnSC . In some embodiments, the cells are contacted with a higher concentration of the HSA (e.g., about 1.0%) in the second period as compared to the first period (e.g., about 0.05%).

In some examples, insulin-positive endocrine cells can be matured in a NS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In some embodiments, the insulin-positive endocrine cells can be matured in a CMRL medium supplemented with 10% FBS. In some embodiments, the insulin-positive endocrine cells can be matured in a DMEM/F12 medium supplemented with 1% HSA. In other cases, SC-P cells can be obtained by culturing the population of cells containing the insulinpositive endocrine cells in a MCDB 131 medium that can be supplemented by 2% BSA. In some embodiments, the MCDB 131 medium with 2% BSA for maturation of insulinpositive endocrine cells into SC-P cells can be comprise no small molecule factors as described herein. In some case, the MCDB131 medium with 2% BSA for maturation of insulin-positive endocrine cells into SC-P cells can comprise no serum (e.g., no FBS). In other cases, SC-P cells can be obtained by culturing the population of cells containing the insulin-positive endocrine cells in a MCDB131 medium that can be supplemented by 0.05% HSA and vitamin C. In some embodiments, SC-P cells can be obtained by culturing the population of cells containing the insulin-positive endocrine cells in a MCDB131 medium that can be supplemented by 0.05% HSA, ITS-X, vitamin C, and glutamine (Gin, e.g., 4mM). In some embodiments, the type of culture medium may be changed during S6. For instance, the S6 cells are cultured in a MCDB131 medium that can be supplemented by 0.05% HSA and vitamin C for the first two to four days, and then followed by a DMEM/F12 medium supplemented with 1% HSA. In some embodiments, additional factors are introduced into the culture medium. For instance, S6 cells can be cultured in a MCDB131 medium that can be supplemented by 0.05% HSA, ITS-X, vitamin C, and glutamine (Gin, e.g., 4mM) throughout the 10-12 days, during which ZnSO4 is introduced from day 4 of S6.

In some embodiments, the medium used to culture the cells as described herein can be xeno-free. A xeno-free medium for culturing cells and/or cell clusters of originated from an animal can have no product from other animals. In some embodiments, a xeno- free medium for culturing human cells and/or cell clusters can have no products from any non-human animals. For example, a xeno-free medium for culturing human cells and/or cell clusters can comprise human platelet lysate (PLT) instead of fetal bovine serum (FBS). For example, a medium can comprise from about 1% to about 20%, from about 5% to about 15%, from about 8% to about 12%, from about 9 to about 11% serum. In some embodiments, medium can comprise about 10% of serum. In some embodiments, the medium can be free of small molecules and/or FBS. For example, a medium can comprise MCDB131 basal medium supplemented with 2% BSA. In some embodiments, the medium is serum-free. In some examples, a medium can comprise no exogenous small molecules or signaling pathway agonists or antagonists, such as, growth factor from fibroblast growth factor family (FGF, such as FGF2, FGF8B, FGF 10, or FGF21), Sonic Hedgehog Antagonist (such as Santl, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol, cyclopamine, or derivatives thereof), Retinoic Acid Signaling agonist (e.g., retinoic acid, CD1530, AM580, TTHPB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, or CD2314), inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK) (e.g., Thiazovivin, Y- 27632, Fasudil/HA1077, or 14-1152), activator of protein kinase C (PKC) (e.g., phorbol 12, 13 -dibutyrate (PDBU) , TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or derivatives thereof), antagonist of TGF P super family (e.g., Alk5 inhibitor II (CAS 446859-33-2), A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, SB-505124, or derivatives thereof), inhibitor of Bone Morphogenetic Protein (BMP) type 1 receptor (e.g., LDN193189 or derivatives thereof), thyroid hormone signaling pathway activator (e.g., T3, GC-1 or derivatives thereof), gamma-secretase inhibitor (e.g., XXI, DAPT, or derivatives thereof), activator of TGF-P signaling pathway (e.g., WNT3a or Activin A) growth factor from epidermal growth factor (EGF) family (e.g., betacellulin or EGF), broad kinase (e.g., staurosporine or derivatives thereof), non-essential amino acids, vitamins or antioxidants (e.g., cyclopamine, vitamin D, vitamin C, vitamin A, or derivatives thereof), or other additions like N- acetyl cysteine, zinc sulfate, or heparin. In some embodiments, the reaggregation medium can comprise no exogenous extracellular matrix molecule. In some embodiments, the reaggregation medium does not comprise Matrigel™. In some embodiments, the reaggregation medium does not comprise other extracellular matrix molecules or materials, such as, collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixtures thereof, for example, or lysed cell membrane preparations.

A person of ordinary skill in the art will appreciate that the concentration of serum albumin supplemented into the medium may vary. For example, a medium (e.g., MCDB131) can comprise about 0.01%, 0.05%, 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% BSA. In other cases, a medium can comprise about 0.01%, 0.05%, 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% HSA. The medium used (e.g., MCDB131 medium) can contain components not found in traditional basal media, such as trace elements, putrescine, adenine, thymidine, and higher levels of some amino acids and vitamins. These additions can allow the medium to be supplemented with very low levels of serum or defined components. The medium can be free of proteins and/or growth factors, and may be supplemented with EGF, hydrocortisone, and/or glutamine. The medium can comprise one or more extracellular matrix molecules (e.g., extracellular proteins). Non-limiting exemplary extracellular matrix molecules used in the medium can include collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin. In some embodiments, the medium comprises laminin, such as LN-332. In some embodiments, the medium comprises heparin.

The medium can be changed periodically in the culture, e.g., to provide optimal environment for the cells in the medium. When culturing the cells dissociated from the first cell cluster for re-aggregation, the medium can be changed at least or about every 4 hours, 12 hours, 24 hours, 48 hours, 3 days or 4 days. For example, the medium can be changed about every 48 hours.

In some embodiments, cells can be cultured under dynamic conditions (e.g., under conditions in which the cells are subject to constant movement or stirring while in the suspension culture). For dynamic culturing of cells, the cells can be cultured in a container (e.g., an non-adhesive container such as a spinner flask (e.g., of 200 ml to 3000 ml, for example 250 ml; of 100 ml; or in 125 ml Erlenmeyer), which can be connected to a control unit and thus present a controlled culturing system. Alternatively, the cells can be cultured in a bioreactor. In some embodiments, cells can be cultured under non-dynamic conditions (e.g., a static culture) while preserving their proliferative capacity. For nondynamic culturing of cells, the cells can be cultured in an adherent culture vessel. An adhesive culture vessel can be coated with any of substrates for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells. The substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used). The substrate for cell adhesion includes collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixtures thereof, for example, Matrigel™, and lysed cell membrane preparations.

Medium in a dynamic cell culture vessel (e.g., a spinner flask or bioreactor) can be stirred (e.g., by a stirrer). The spinning speed can correlate with the size of the reaggregated second cell cluster. The spinning speed can be controlled so that the size of the second cell cluster can be similar to an endogenous pancreatic islet. In some embodiments, the spinning speed is controlled so that the size of the second cell cluster can be from about 75 pm to about 250 pm. The spinning speed of a dynamic cell culture vessel (e.g., a spinner flask or bioreactor) can be about 20 rounds per minute (rpm) to about 100 rpm, e.g., from about 30 rpm to about 90 rpm, from about 40 rpm to about 60 rpm, from about 45 rpm to about 50 rpm. In some embodiments, the spinning speed can be about 50 rpm.

Stage 6 cells as provided herein may or may not be subject to the dissociation and reaggregation process as described herein. In some embodiments, the cell cluster comprising the insulin-positive endocrine cells can be reaggregated. The reaggregation of the cell cluster can enrich the insulin-positive endocrine cells. In some embodiments, the insulin-positive endocrine cells in the cell cluster can be further matured into pancreatic P cells. For example, after reaggregation, the second cell cluster can exhibit in vitro GSIS, resembling native pancreatic islet. For example, after reaggregation, the second cell cluster can comprise non-native pancreatic P cell that exhibits in vitro GSIS. In some embodiments, the reaggregation process can be performed according to the disclosure of PCT application PCT/US2018/043179, which is incorporated herein by reference in its entirety.

Stage 6 cells obtained according to methods provided herein can have high recovery yield after cryopreservation and reaggregation procedures. In some embodiments, stage 6 cells that are obtained in a differentiation process that involves treatment of a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-P superfamily (e.g., Activin A) at stage 3 and treatment of an epigenetic modifying compound (e.g., histone methyltransferase inhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have a higher recovery yield after cry opreservation post stage 5, as compared to a corresponding cell population without such treatment. In some embodiments, stage 6 cells that are obtained in a differentiation process that involves treatment of a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-P superfamily (e.g., Activin A) at stage 3 and treatment of an epigenetic modifying compound (e.g., histone methyltransferase inhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have a higher recovery yield after cry opreservation post stage 5, as compared to a corresponding cell population without treatment of a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-P superfamily (e.g., Activin A) at stage 3. In some embodiments, stage 6 cells that are obtained in a differentiation process that involves treatment of a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-P superfamily (e.g., Activin A) at stage 3 and treatment of an epigenetic modifying compound (e.g., histone methyltransferase inhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have a recovery yield after cryopreservation post stage 5 that is at least about 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 48%, 49%, or 50%. The recovery yield can be calculated as a percentage of cells that survive and form reaggregated cell clusters after cryopreservation, thawing and recovery, and reaggregation procedures, as compared to the cells before the cryopreservation.

In some embodiments, the present disclosure relates to cry opreservation of the non-native pancreatic P cells or precursors thereof obtained using the methods provided herein. In particular embodiments, the cells are cryopreserved following stage 5 and before stage 6. In some embodiments, the cell population comprising non-native pancreatic P cells can be stored via cryopreservation. For instances, the cell population comprising non-native P cells, e.g., Stage 6 cells are thawed. In some embodiments, the cells can be dissociated into cell suspension, e.g., single cell suspension, and the cell suspension can be cryopreserved, e.g., frozen in a cry opreservation solution. The dissociation of the cells can be conducted by any of the technique provided herein, for example, by enzymatic treatment. The cells can be frozen at a temperature of at highest - 20 °C, at highest -30 °C, at highest -40 °C, at highest -50 °C, at highest -60 °C, at highest - 70 °C, at highest -80 °C, at highest -90 °C, at highest -100 °C, at highest -110 °C, at highest -120 °C, at highest -130 °C, at highest -140 °C, at highest -150 °C, at highest -160 °C, at highest -170 °C, at highest -180 °C, at highest -190 °C, or at highest -200 °C. In some embodiments, the cells are frozen at a temperature of about -80 °C. In some embodiments, the cells are frozen at a temperature of about -195 °C. Any cooling methods can be used for providing the low temperature needed for cry opreservation, such as, but not limited to, electric freezer, solid carbon dioxide, and liquid nitrogen. In some embodiments, any cry opreservation solution available to one skilled in the art can be used for incubating the cells for storage at low temperature, including both custom made and commercial solutions. For example, a solution containing a cryoprotectant can be used. The cryoprotectant can be an agent that is configured to protect the cell from freezing damage. For instance, a cryoprotectant can be a substance that can lower the glass transition temperature of the cryopreservation solution. Exemplary cryoprotectants that can be used include DMSO (dimethyl sulfoxide), glycols (e.g., ethylene glycol, propylene glycol and glycerol), dextran (e.g., dextran-40), and trehalose. Additional agents can be added into the cryopreservation solution for other effects. In some embodiments, commercially available cry opreservation solutions can be used in the method provided herein, for instance, FrostaLife™, pZerve™, Prime-XV®, Gibco Synth-a-Freeze Cryopreservation Medium, STEM-CELLB ANKER®, CryoStor® Freezing Media, HypoThermosol® FRS Preservation Media, and CryoDefend® Stem Cells Media.

During the differentiation process, the cells can be subject to irradiation treatment as provided herein. In some embodiments, the cell population at Stage 6, e.g., the cell population or cell cluster that has cells being differentiated from insulin-positive endocrine cells into pancreatic P cells, is irradiated for a period of time. In some embodiments, the cell population at Stage 6 after reaggregation following the recovery from cryopreservation is irradiated for a period of time. In some embodiments, the cryopreserved cells (e.g., the cells that are cryopreserved at the end of Stage 5) are irradiated for a certain period of time prior to thawing and recovery for subsequent differentiation process.

In some embodiments, the stage 6 cells comprise NKX6.1 -positive, insulinpositive cells. In some embodiments, the stage 6 cells comprise NKX6.1 -positive, insulinnegative cells. In some embodiments, the stage 6 cells comprise C-peptide positive cells. In some embodiments, Stage 6 cells or cells that have characteristics of stage 6 cells are incubated in NS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In some embodiments, the stage 6 cells or cells that have characteristics of stage 6 cells are contacted with any one or more of a vitamin or anti-oxidant (e.g., vitamin C), an albumin protein (e.g., a human serum albumin protein), a TGF-beta pathway inhibitor (e.g., an ALK5 inhibitor II), a bone morphogenic protein (BMP) type 1 receptor inhibitor (e.g., LDN193189), a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin), a histone methyltransferase inhibitor (e.g., DZNEP), and a protein kinase inhibitor (e.g., staurosporine). See, e.g.., W02020264072. In some embodiments, the stage 6 cells are contacted with a PKC activator (see, e.g., WO2019217487, which is incorporated by reference herein in its entirety).

Differentiation factors

Aspects of the disclosure relate to contacting progenitor cells (e.g., stem cells, e.g., iPS cells, definitive endoderm cells, primitive gut tube cells, PDX1 -positive pancreatic progenitor cells, NKX6.1 -positive pancreatic progenitor cells, insulin-positive endocrine cells) with p cell differentiation factors, for example, to induce the maturation of the insulin-positive endocrine cells or differentiation of other progenitor cells into SC-P cells (e.g., mature pancreatic P cells). In some embodiments, the differentiation factor can induce the differentiation of pluripotent cells e.g., iPSCs or hESCs) into definitive endoderm cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor can induce the differentiation of definitive endoderm cells into primitive gut tube cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor(s) can induce the differentiation of primitive gut tube cells into PDX1 -positive pancreatic progenitor cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor(s) can induce the differentiation of PDX1 -positive pancreatic progenitor cells into NKX6-1- positive pancreatic progenitor cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor(s) can induce the differentiation of NKX6-1- positive pancreatic progenitor cells into insulin-positive endocrine cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor(s) can induce the maturation of insulin-positive endocrine cells into pancreatic islet cells, e.g., in accordance with a method described herein.

At least one differentiation factor described herein can be used alone, or in combination with other differentiation actors, to generate pancreatic islet cells (e.g., SC- beta cells) according to the methods as disclosed herein. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten differentiation factors described herein are used in the methods of generating pancreatic islet cells.

In some embodiments, a composition described herein does not comprise one or more of the differentiation factors provided herein.

Amino acids

Aspects of the disclosure relate to the use of culture media supplemented with additional amino acids for differentiation. As described herein, the term “amino acid” may broadly refer to compounds containing both a carboxyl group and an amino group and may refer to an amino acid in its many different chemical forms including a single administration amino acid, its physiologically active salts or esters, its combinations with its various salts, its tautomeric, polymeric and/or isomeric forms, its analog forms, its derivative forms, its products of biosynthesis, and/or its decarboxylation products. Amino acids may describe both essential amino acids and/or non-essential amino acids. As described herein, an “essential amino acid” may refer to an amino acid that cannot be

I l l made by the body and is consumed through diet. In some embodiments essential amino acids may include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. As described herein, a “non-essential amino acid” may refer to an amino acid that can be made by the body and does not need to be obtained directly through dietary intake. In some embodiments, non-essential amino acids may include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine.

In some embodiments, in a method described herein comprises a medium supplemented with additional amino acids including aspartate, glycine or serine, or combinations thereof. As described herein, “aspartate” or “aspartic acid” may refer to a non-essential amino acid that has a side chain (CH2COOH). Aspartate may be present in two forms, or enantiomers. These two forms may include either D-aspartic acid or L- aspartic acid. In some embodiments, aspartic acid may be present in a racemic mixture “DL-aspartic acid”. As described herein, “glycine” may refer to an amino acid that has a single hydrogen atom as its side chain. As described herein, “serine” may refer to an- amino acid that has a side chain of a hydroxymethyl group.

PI3K/Akt/mTOR Inhibitor

Aspects of the disclosure relate to the use of PI3K/Akt/mT0R signaling inhibitors as differentiation factors. As described herein, the term “PI3K” may refer to the phosphatidylinositol 3 -kinases, which may refer to a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking phosphatidylinositol-3-kinase. PI3Ks may also refer to intracellular signal transducer enzymes capable of phosphorylating the 3 -position hydroxyl group of the inositol ring of phosphatidylinositol. As described herein, the term “Akt” may refer to a family of genes that encode isoforms of Protein kinase B, sometimes referred to as AKT1, AKT2 and AKT3 and encode the RAC alpha, beta, and gamma serine/threonine protein kinases respectively. In some embodiments, Akt may refer to the products of all three genes collectively, or individually. As described herein, the term “mTOR” may refer to mammalian target of rapamycin, mechanistic target of rapamycin, FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1) or a member of the phosphatidylinositol 3 -kinase-related kinase family of protein kinases. In some embodiments, mTOR may describe a protein that serves as a core component of two protein complexes, mTOR complex 1 and mTOR complex 2. In some embodiments, mTOR functions as a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription, mTOR also functions as a tyrosine protein kinase that promotes the activation of insulin receptors and insulin-like growth factor 1 receptors and the control and maintenance of the actin cytoskeleton

As described herein, the term “PI3K/Akt/mT0R signaling” may refer to an intracellular signaling pathway involving any of the following component alone or in combination” PI3K, Akt or mTOR. In some embodiments “PI3K/Akt/mT0R signaling may be involved in regulating the cell cycle or the response to cellular stress.

In some embodiments, in a method described herein comprises a medium comprising an inhibitor of PI3K/Akt/mT0R signaling (e.g., GSK-690693). In some embodiments, the inhibitor of PI3K/Akt/mT0R signaling may be selected from, but is not limited to, one or more of: alpelisib (BYL719), idelalisib, copanlisib, buparlisib (BKM120) and pictilisib (GDC-0941), taselisib (GDC-0032), and BEZ235, ipatasertib (GDC-0068), capivasertib (AZD-5363), everolimus, temsirolimus (CCI-779), GSK- 690693, IPI-3063, AZD8055, Omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, Nemiralisib, BYL719, FT113, or Apitolisib, or any analog or derivative thereof. In some embodiments, the inhibitor of PI3K/Akt/mT0R signaling is GSK-690693 or an analog or a derivative thereof.

Forkhead Box 01 (FoxOl) inhibitor

Aspects of the disclosure relate to the use of Forkhead Box 01 (FoxOl) inhibitors as differentiation factors. In some embodiments, the FoxOl inhibitor used in the compositions and methods described herein is a compound of Formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, prodrug, composition, or mixture thereof, wherein: R¹ is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group;

R² is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; each instance of R³ is independently optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R³ are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring; each instance of R⁴ is independently halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, - OR^C1, -NO₂, -N(R^C2)₂, -SR^C1, -CN, or -SCN;

R⁵ is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; each instance of R⁶ is independently halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, - OR^C1, -NO2, -N(R^C2)₂, -SR^C1, -CN, or -SCN; wherein R^C1 is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; wherein each instance of R^c2 is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R^c2 are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring; x is 0, 1, or 2; y is 0 or 1; and z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as valency permits.

In some embodiments, the compound is of Formula (I-A):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, prodrug, composition, or mixture thereof, wherein:

R¹ is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R² is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; each instance of R³ is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R⁴ is halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl; and

R⁵ is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In some embodiments, R¹ is hydrogen. In some embodiments, R² is optionally substituted alkyl. In some embodiments, R² is ethyl. In some embodiments, at least one instance of R³ is hydrogen. In some embodiments, both instances of R³ are hydrogen. In some embodiments, at least one instance of R⁴ is halogen. In some embodiments, at least one instance of R⁴ is fluorine. In some embodiments, x is 1. In some embodiments, R⁵ is hydrogen. In some embodiments, y is 1. In some embodiments, z is 0.

In some embodiments, the compound is of formula:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, prodrug, composition, or mixture thereof.

In some embodiments, the compound is AS 1842856.

In some embodiments, a medium described herein does not comprise a FoxOl inhibitor.

Transforming Growth Factor -fl (IGF- fl) Superfamily

Aspects of the disclosure relate to the use of growth factors from the transforming growth factor-P (TGF-P) superfamily as differentiation factors. The “TGF-P superfamily” means proteins having structural and functional characteristics of known TGFP family members. The TGFP family of proteins can include the TGFP series of proteins, the Inhibins (including Inhibin A and Inhibin B), the Activins (including Activin A, Activin B, and Activin AB), MIS (Mullerian inhibiting substance), BMP (bone morphogenetic proteins), dpp (decapentaplegic), Vg-1, MNSF (monoclonal nonspecific suppressor factor), and others. Activity of this family of proteins can be based on specific binding to certain receptors on various cell types. Members of this family can share regions of sequence identity, particularly at the C-terminus, that correlate to their function. The TGFP family can include more than one hundred distinct proteins, all sharing at least one region of amino acid sequence identity. Members of the family that can be used in the method disclosed herein can include, but are not limited to, the following proteins, as identified by their GenBank accession numbers: P07995, Pl 8331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917, P09529, P27093, P04088, Q04999, P17491, P55104, Q9WUK5, P55103, 088959, 008717, P58166, 061643, P35621, P09534, P48970, Q9NR23, P25703, P30884, P12643, P49001, P21274, 046564, 019006, P22004, P20722, Q04906, Q07104, P30886, P18075, P23359, P22003, P34821, P49003, Q90751, P21275, Q06826, P30885, P34820, Q29607, P12644, Q90752, 046576, P27539, P48969, Q26974, P07713, P91706, P91699, P27091, 042222, Q24735, P20863, 018828, P55106, Q9PTQ2, 014793, 008689, 042221, 018830, 018831, 018836, 035312, 042220, P43026, P43027, P43029, 095390, Q9R229, 093449, Q9Z1W4, Q9BDW8, P43028, Q7Z4P5, P50414, P17246, P54831, P04202, P01137, P09533, P18341, 019011, Q9Z1Y6, P07200, Q9Z217, 095393, P55105, P30371, Q9MZE2, Q07258, Q96S42, P97737, AAA97415.1, NP-776788.1, NP-058824.1, EAL24001.1, 1 S4Y, NP-001009856.1, NP-1- 032406.1, NP-999193.1, XP-519063.1, AAG17260.1, CAA40806.1, NP- 1-001009458.1, AAQ55808.1, AAK40341.1, AAP33019.1, AAK21265.1, AAC59738.1, CAI46003.1, B40905, AAQ55811.1, AAK40342.1, XP-540364.1, P55102, AAQ55810.1, NP-

990727.1, CAA51163.1, AAD50448.1, JC4862, PN0504, BAB17600.1, AAH56742.1, BAB17596.1, CAG06183.1, CAG05339.1, BAB17601.1, CAB43091.1, A36192, AAA49 162.1, AAT42200.1, NP-789822.1, AAA59451.1, AAA59169.1, XP-541000.1, NP-990537.1, NP- 1-002184.1, AAC14187.1, AAP83319.1, AAA59170.1, BAB16973.1, AAM66766.1, WFPGBB, 1201278C, AAH30029.1, CAA49326.1, XP-344131.1, AA-

148845.1, XP-1-148966.3, 148235, B41398, AAH77857.1, AAB26863.1, 1706327A, BAA83804.1, NP-571143.1, CAG00858.1, BAB17599.1, BAB17602.1, AAB61468.1, PN0505, PN0506, CAB43092.1, BAB17598.1, BAA22570.1, BAB16972.1, BAC81672.1, BAA12694.1, BAA08494.1, B36192, C36192, BAB16971.1, NP-034695.1, AAA49160.1, CAA62347.1, AAA49161.1, AAD30132.1, CAA58290.1, NP-005529.1, XP-522443.1, AAM27448.1, XP-538247.1, AAD30133. I, AAC36741.1, AAH10404.1, NP-032408.1, AAN03682.1, XP-509161.1, AAC32311.1, NP-651942.2, AAL51005.1, AAC39083.1, AAH85547.1, NP-571023.1, CAF94113.1, EAL29247.1, AAW30007.1, AAH90232.1, A29619, NP-001007905.1, AAH73508.1, AADO2201.1, NP-999793.1, NP-990542.1, AAF19841.1, AAC97488.1, AAC60038.1, NP 989197.1, NP-571434.1, EAL41229.1, AAT07302.1, CAI19472.1, NP-031582.1, AAA40548.1, XP-535880.1, NP-1-037239.1, AAT72007.1, XP-418956.1, CAA41634.1, BAC30864.1, CAA38850.1, CAB81657.2, CAA45018.1, CAA45019.1, BAC28247.1, NP-031581.1, NP-990479.1, NP-999820.1, AAB27335.1, S45355, CAB82007.1, XP-534351.1, NP-058874.1, NP-031579.1, 1REW, AAB96785.1, AAB46367.1, CAA05033.1, BAA89012.1, IES7, AAP20870.1, BAC24087.1, AAG09784.1, BAC06352.1, AAQ89234.1, AAM27000.1, AAH30959.1, CAGO1491.1, NP-571435.1, 1REU, AAC60286.1, BAA24406.1, A36193, AAH55959.1, AAH54647.1, AAH90689.1, CAG09422.1, BAD16743.1, NP-032134.1, XP-532179.1, AAB24876.1, AAH57702.1, AAA82616.1, CAA40222.1, CAB90273.2, XP-342592.1, XP-534896.1, XP-534462.1, 1LXI, XP-417496.1, AAF34179.1, AAL73188.1, CAF96266.1, AAB34226.1, AAB33846.1, AAT12415.1, AA033819.1, AAT72008.1, AAD38402.1, BAB68396.1, CAA45021.1, AAB27337.1, AAP69917.1, AATI2416.1, NP-571396.1, CAA53513.1, AA033820.1, AAA48568.1, BAC02605.1, BAC02604.1, BAC02603.1, BAC02602.1, BAC02601.1, BAC02599.1, BAC02598.1, BAC02597.1, BAC02595.1, BAC02593.1, BAC02592.1, BAC02590.1, AAD28039.1, AAP74560.1, AAB94786.1, NP-001483.2, XP-528195.1, NP-571417.1, NP-001001557. 1, AAH43222.1, AAM33143.1, CAG10381.1, BAA31132.1, EAL39680.1, EAA12482.2, P34820, AAP88972.1, AAP74559.1, CAI16418.1, AAD30538.1, XP-345502.1, NP-1- 038554.1, CAG04089.1, CAD60936.2, NP-031584.1, B55452, AAC60285.1, BAA06410.1, AAH52846.1, NP-031580.1, NP-1-036959.1, CAA45836.1, CAA45020.1, Q29607, AAB27336.1, XP-547817.1, AAT12414.1, AAM54049.1, AAH78901.1, AA025745.1, NP-570912.1, XP-392194.1, AAD20829.1, AAC97113.1, AAC61694.1, AAH60340.1, AAR97906.1, BAA32227.1, BAB68395.1, BAC02895.1, AAWS 1451.1, AAF82188.1, XP-544189.1, NP-990568.1, BAC80211.1, AAW82620.1, AAF99597.1, NP-571062.1, CAC44179.1, AAB97467.1, AAT99303.1, AAD28038.1, AAH52168.1, NP-001004122.1, CAA72733.1, NP-032133.2, XP-394252.1, XP-224733.2, JH0801, AAP97721.1, NP -989669.1, S43296, P43029, A55452, AAH32495.1, XP-542974.1, NP- 032135.1, AAK30842.1, AAK27794.1, BAC30847.1, EAA12064.2, AAP97720.1, XP- 525704.1, AAT07301.1, BAD07014.1, CAF94356.1, AAR27581.1, AAG13400.1, AAC60127.1, CAF92055.1, XP-540103.1, AA020895.1, CAF97447.1, AAS01764.1, BAD08319.1, CAA10268.1, NP-998140.1, AAR03824.1, AAS48405.1, AAS48403.1, AAK53545.1, AAK84666.1, XP-395420.1, AAK56941.1, AAC47555.1, AAR88255.1, EAL33036.1, AAW47740.1, AAW29442.1, NP-722813.1, AARO8901.1, AAO 15420.2, CAC59700.1, AAL26886.1, AAK71708.1, AAK71707.1, CAC51427.2, AAK67984.1, AAK67983.1, AAK28706.1, P07713, P91706, P91699, CAG02450.1, AAC47552.1, NP- 005802.1, XP-343149.1, AW34055.1, XP-538221.1, AAR27580.1, XP-125935.3, AAF21633.1, AAF21630.1, AAD05267.1, Q9Z1 W4, NP-1-031585.2, NP-571094.1, CAD43439.1, CAF99217.1, CAB63584.1, NP-722840.1, CAE46407.1, XP- 1-417667.1, BAC53989.1, BAB19659.1, AAM46922.1, AAA81169.1, AAK28707.1, AAL05943.1, AAB 17573.1, CAH25443.1, CAG10269.1, BAD 16731.1, EAA00276.2, AAT07320.1, AAT07300.1, AAN15037.1, CAH25442.1, AAK08152.2, 2009388A, AAR12161.1, CAGO1961.1, CAB63656.1, CAD67714.1, CAF94162.1, NP-477340.1, EAL24792.1, NP- 1-001009428.1, AAB86686.1, AAT40572.1, AAT40571.1, AAT40569.1, NP- 033886.1, AAB49985.1, AAG39266.1, Q26974, AAC77461.1, AAC47262.1, BAC05509.1, NP -055297.1, XP-546146.1, XP-525772.1, NP-060525.2, AAH33585.1, AAH69080.1, CAG12751.1, AAH74757.2, NP-034964.1, NP-038639.1, 042221, AAF02773.1, NP-062024.1, AAR18244.1, AAR14343.1, XP-228285.2, AAT40573.1, AAT94456.1, AAL35278.1, AAL35277.1, AAL17640.1, AAC08035.1, AAB86692.1, CAB40844.1, BAC38637.1, BAB16046.1, AAN63522.1, NP-571041.1, AAB04986.2, AAC26791.1, AAB95254.1, BAA11835.1, AAR18246.1, XP-538528.1, BAA31853.1, AAK18000.1, XP-1-420540.1, AAL35276.1, AAQ98602.1, CAE71944.1, AAW50585.1, AAV63982.1, AAW29941.1, AAN87890.1, AAT40568.1, CAD57730.1, AAB81508.1, AAS00534.1, AAC59736.1, BAB79498.1, AAA97392.1, AAP85526.1, NP-999600.2, NP-878293.1, BAC82629.1, CAC60268.1, CAG04919.1, AAN10123.1, CAA07707.1 AAK20912.1, AAR88254.1, CAC34629.1, AAL35275.1, AAD46997. I, AAN03842.1, NP-571951.2, CAC50881.1, AAL99367.1, AAL49502.1, AAB71839.1, AAB65415.1, NP-624359.1, NP-990153.1, AAF78069.1, AAK49790.1, NP-919367.2, NP-001192.1, XP-544948.1, AAQ18013.1, AAV38739.1, NP-851298.1, CAA67685.1, AAT67171.1, AAT37502.1, AAD27804.1, AAN76665.1, BAC11909.1, XP-1-421648.1, CAB63704.1, NP-037306.1, A55706, AAF02780.1, CAG09623.1, NP-067589.1, NP-035707.1, AAV30547.1, AAP49817.1, BAC77407.1, AAL87199.1, CAG07172.1, B36193, CAA33024.1, NP- 1-001009400.1, AAP36538.1, XP-512687.1, XP-510080.1, AAH05513.1, 1KTZ, AAH14690.1, AAA31526.1.

The growth factor from the TGF-P superfamily in the methods and compositions provided herein can be naturally obtained or recombinant. In some embodiments, the growth factor from the TGF-P superfamily comprises Activin A. The term “Activin A” can include fragments and derivatives of Activin A. The sequence of an exemplary Activin A is provided as SEQ ID NO: 1. Other non-limiting examples of Activin A are provided in SEQ ID NO: 3-16, and non-limiting examples of nucleic acids encoding Activin A are provided in SEQ ID NO: 2, SEQ ID NO: 17, and SEQ ID NO: 18 . In some embodiments, the growth factor from the TGF-P superfamily comprises a polypeptide comprising an amino acid sequence that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 3-16, or functional fragments thereof. In some embodiments, the growth factor from the TGF-P superfamily comprises a polypeptide comprising the amino acid any one of SEQ ID NOs: 1 and 3-16.

SEQ ID NO: 1 - Homo sapiens Inhibin beta A subunit (Activin A) amino acid sequence:

GLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSL SFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVE ECGCS

SEQ ID NO: 2 - Homo sapiens Inhibin beta A chain (Activin A) nucleic acid sequence: GGCTTGGAGTGTGATGGCAAGGTCAACATCTGCTGTAAGAAACAGTTCTTTGT CAGTTTCAAGGACATCGGCTGGAATGACTGGATCATTGCTCCCTCTGGCTATC ATGCCAACTACTGCGAGGGTGAGTGCCCGAGCCATATAGCAGGCACGTCCGG GTCCTCACTGTCCTTCCACTCAACAGTCATCAACCACTACCGCATGCGGGGCC ATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTGCCCACCAAGCTGAGACCC ATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCA GAACATGATCGTGGAGGAGTGTGGGTGCTCATAG

SEQ ID NO: 3 - Homo sapiens Erythroid differentiation protein (EDF) ovarian amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVE AVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRR

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANR TRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVF P VS S SIQRLLDQGKS SLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGE GGAGADEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSF KDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFAN LKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 4 - Homo sapiens Inhibin B subunit amino acid sequence:

ARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCE GECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDD

GQNIIKKDIQNMIVEECGCS SEQ ID NO: 5 - Homo sapiens Inhibin B subunit in testis Homo sapiens amino acid sequence:

GLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSL SFHSTVINHYACGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEEC GCS

SEQ ID NO: 6 - Homo sapiens Inhibin B subunit erythroid differentiation protein (EDF), amino acid sequence:

SEQ ID NO: 7 - Mus musculus (Mouse) Inhibin beta A chain (Activin beta- A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHGSAPDCPSCALATLPKDGPNSQPEMVE AVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRR

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANR TRTKVTIRLFQQQKHPQGSLDTGDEAEEMGLKGERSELLLSEKVVDARKSTWHIF P VS S SIQRLLDQGKS SLDVRIACEQCQESGASLVLLGKKKKKEVDGDGKKKDGSD GGLEEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFKDI

GWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 8 - Rattus norvegicus (Rat) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHGAAPDCPSCALATLPKDGPNSQPEMVE AVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRR

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANR TRTKVTIRLFQQQKHPQGSLDMGDEAEEMGLKGERSELLLSEKVVDARKSTWHIF P VS S SIQRLLDQGKS SLDVRIACEQCQESGASLVLLGKKKKKEVDGDGKKKDGSD GGLEEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFKDI GWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 9 - Gallus gallus (Chicken) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWKRGFLLVICWIIVRSSPTPGSEGHSSVADCPSCALTTLSKDVPSSQPEMVE AVKKHILNMLHLRDRPNITQPVPKAALLNATKKLHVGKVGDDGYVEIEDDVGRR

AEMNEVVEQTSEIITFAESGTPKKTLHFEISKEGSELSVVEHAEVWLFLKVSKANR SRTKVTIRLFQQQRQPKGNSEAAEDMEDMGLKGERSETLISEKAVDARKSTWHIF PIS S S VQRLLDQGQ S SLD VRIACDLCQETGASLVLLGKKKKKEDDGEGKEKDGGE LTGEEEKEQSHRPFLMMLARHSEDRQHRRRERGLECDGKVNICCKKQFFVSFKDI GWSDWIIAPTGYHANYCEEECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 10 - Bos taurus (Bovine) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALATLPKDVPNSQPEMVE AVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRR AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEIWLFLKVPKANRT RSKVTIRLFQQQKHLQGSLDAGEEAEEVGLKGEKSEMLISEKVVDARKSTWHIFP VS SCIQRLLDQGKS SLDIRIACEQCQETGASLVLLGKKKKKEEEGEGKKRDGEGG AGGDEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFKDI GWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 11 - Equus caballus (Horse) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVKSSPTPGSEGHSAAPNCPSCALATLPKDVPNAQPEMVE AVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRR AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANR TRSKVTIRLLQQQKHPQGSSDTREEAEEADLMEERSEQLISEKVVDARKSTWHIFP VS S SIQRLLDQGKS SLDIRIACDQCHETGASLVLLGKKKKKEEEGEGKKKDGGEA GAGVDEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFK DIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINQYRLRGHNPFANL KSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS SEQ ID NO: 12 - Sus scrofa (Pig) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSGGHSAAPDCPSCALATLPKDVPNSQPEMVE AVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVELEDDIGRR AEMNELMEQTSEIITFAEAGTARKTLRFEISKEGSDLSVVERAEIWLFLKVPKANR TRTKVSIRLFQQQRRPQGSADAGEEAEDVGFPEEKSEVLISEKVVDARKSTWHIFP VS S SIQRLLDQGKS ALDIRT ACEQCHETGASLVLLGKKKKKEEEAEGRKRDGEGA GVDEEKEQSHRPFLMLQARQSEEHPHRRRRRGLECDGKVNICCKKQFFVSFKDIG WNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSC CVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 13 - Ovis aries (Sheep) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALATLPKDVPNSQPEMVE AVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRR AEMNELMEQTSEIITFAESGTARKTLHFEISQEGSDLSVVERAEIWLFLKVPKANRT RSKVTIRLFQQQKHLQGSLDAGEEAEEVGLKGEKSEMLISEKVVDARKSTWHIFP VS SCIQRLLDQGKS SLDIRIACEQCQETGASLVLLGKKKRKEEEGEGKKRDGEGG AGGDEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQFYVSFKDI GWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 14 - Felis catus (cat) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGPGAAPDCPSCALATLPKDVPNSQPEMVE AVKKHILNMLHLKKRPEVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRA EMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRT RTKVTIQLLQKQPQGGVDAGEEAEEMGLMEERNEVLISEKVVDARKSTWHIFPVS S SIQRLLDQGKS SLDVRIACEQCHETGASLVLLGKKKKKEEEGEGKKKDGGDGG AGADEDKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFKDI GWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 15 - Danio rerio (zebrafish) Inhibin beta A chain (Activin beta-A chain) amino acid sequence: MSPLPLLSGILLLLIRSCSLSAMVTKGSLPMSEQQAGATVCPSCALARFRKGVSESE DEGAQQDVVEAVI<RHILNMLHLQERPNITHPVPRAALLNAIRI<VHVGRVAI<DGS VLIEDEASNRAETEQAEQTEIITFAETGEAPGIVNFLISKEGGEMSVVDQANVWIFL RLPKGNRTRANVNIRLLLQQGAGEKILAEKSVDTRRSGWHTFPASESVQSLLQRG GSTLSLRVSCPLCADARATPVLVSPGGSEREQSHRPFLMAVVRQMDELSLRRRRK RGLECDGKARVCCKRQFYVNFKDIGWNDWIIAPSGYHANYCEGDCASNVASITG NSLSFHSTVISHYRIRGYSPFTNIKSCCVPTRLRAMSMLYYNEEQKIVKKDIQNMIV EECGCS

SEQ ID NO: 16 - Carassius auratus (goldfish) Inhibin beta A chain (Activin beta-A chain) amino acid sequence: MSSLTLVNRGTAALRLFVRGLLTHSSREWLSGDGEPDDPVTPCPSCALAQRQKDS EEQTDMVEAVKRHILNMLHLNTRPNVTHPVPRAALLNAIRRLHVGRVGEDGTVE MEEDGGGLGEHREQSEEQPFEIITFAEPGDAPDIMKFDISMEGNTLSVVEQANVWL LLKVAKGSRGKGKVSVQLLQHGKADPGSADGPQEAVVSEKTVDTRRSGWHTLP VSRTVQTLLDGDSSMLSLRVSCPMCAEAGAVPILVPTESNKGKEREQSHRPFLMV VLKPAEEHPHRRSKRGLECDGKIRVCCKRQFYVNFKDIGWSDWIIAPSGYHANYC EGDCPSHVASITGSALSFHSTVINHYRMRGYSPFNNIKSCCVPTRLRAMSMLYYNE

EQKIIKKDIQNMIVEECGC S

SEQ ID NO: 17 - Recombinant Inhibin B subunit nucleic acid sequence GCCCGGCAGTCTGAAGACCACCCTCATCGCCGGCGTCGGCGGGGCTTGGAGTG TGATGGCAAGGTCAACATCTGCTGTAAGAAACAGTTCTTTGTCAGTTTCAAGG ACATCGGCTGGAATGACTGGATCATTGCTCCCTCTGGCTATCATGCCAACTAC TGCGAGGGTGAGTGCCCGAGCCATATAGCAGGCACGTCCGGGTCCTCACTGTC CTTCCACTCAACAGTCATCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAAC CTCAAATCGTGCTGTGTGCCCACCAAGCTGAGACCCATGTCCATGTTGTACTATGATG ATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTGGAGGAGTGTGGGT GCTCATAGAGTTGCCCAGCCCAGGGGGAAAGGGAGCAAGA

SEQ ID NO : 18 - Homo sapiens mature subunit beta(A) inhibin in testis nucleic acid sequence GGCCTGGAGTGCGACGGCAAGGTCAACATCTGCTGTAAGAAACAGTTCTTTGT CAGTTTCAAGGACATCGGCTGGAATGACTGGATCATTGCTCCCTCTGGCTATC ATGCCAACTACTGCGAGGGTGAGTGCCCGAGCCATATAGCAGGCACGTCCGG GTCCTCACTGTCCTTCCACTCAACAGTCATCAACCACTACGCATGCGGCCATA GCCCCTTTGCCAACCTCAAATCGTGCTGTGTGCCCACCAAGCTGAGACCCATG TCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAA CATGATCGTGGAGGAGTGCGGGTGCTCCTAA

In some embodiments, the growth factor from the TGF-P superfamily comprises growth differentiation factor 8 (GDF8). The term “GDF8” can include fragments and derivatives of GDF8. The sequences of GDF8 polypeptides are available to the skilled artisan. In some embodiments, the growth factor from the TGF-P superfamily comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human GDF8 polypeptide sequence (GenBank Accession EAX10880).

In some embodiments, the growth factor from the TGF-P superfamily comprises a growth factor that is closely related to GDF8, e.g., growth differentiation factor 11 (GDF11). In some embodiments, the growth factor from the TGF-P superfamily comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human GDF11 polypeptide sequence (GenBank Accession AAF21630).

In some embodiments, the growth factor from the TGF-P superfamily can be replaced with an agent mimics the at least one growth factor from the TGF-P superfamily. Exemplary agents that mimic the at least one growth factor from the TGF-P superfamily, include, without limitation, IDE1 and IDE2.

In some embodiments, a medium described herein does not comprise a TGF-P superfamily protein.

Bone Morphogenetic Protein (BMP) Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of BMP signaling pathway inhibitors (which also may be referred to as “BMP inhibitors” herein) as cell differentiation factors. The BMP signaling family is a diverse subset of the TGF-P superfamily (Sebald et al. Biol. Chem. 385:697-710, 2004). Over twenty known BMP ligands are recognized by three distinct type II (BMPRII, ActRIIa, and ActRIIb) and at least three type I (ALK2, ALK3, and ALK6) receptors. Dimeric ligands facilitate assembly of receptor heteromers, allowing the constitutively-active type II receptor serine/threonine kinases to phosphorylate type I receptor serine/threonine kinases. Activated type I receptors phosphorylate BMP -responsive (BR-) SMAD effectors (SMADs 1, 5, and 8) to facilitate nuclear translocation in complex with SMAD4, a co-SMAD that also facilitates TGF signaling. In addition, BMP signals can activate intracellular effectors such as MAPK p38 in a SMAD-independent manner (Nohe et al. Cell Signal 16:291-299, 2004). Soluble BMP antagonists such as noggin, chordin, gremlin, and folli statin limit BMP signaling by ligand sequestration.

In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises DMH-1, or a derivative, analogue, or variant thereof. In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises the following compound or a derivative, analogue, or variant of the following compound:

In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises LDN193189 (also known as LDN193189, 1062368-24-4, LDN-193189, DM 3189, DM-3189, IUPAC Name: 4-[6-(4-piperazin-l- ylphenyl)pyrazolo[l,5-a]pyrimidin-3-yl]quinolone). In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises the following compound or a derivative, analogue, or variant of the following compound:

In some embodiments, DMH-1 can be more selective as compared to LDN193189. In some embodiments of the present disclosure, DMH-1 can be particularly useful for the methods provided herein. In some embodiments, the methods and compositions provided herein, or specific stages of the methods disclosed herein (e.g., stage 3), exclude use of LDN193189. In some embodiments, the methods and compositions provided herein exclude use of LDN 193189, or a derivative, analogue, or variant thereof for generating PDX1 -positive pancreatic progenitor cells from primitive gut tube cells. In some embodiments, the methods and compositions provided herein relate to use of DMH-1, or a derivative, analogue, or variant thereof for generating PDX1 -positive pancreatic progenitor cells from primitive gut tube cells.

In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprise an analog or derivative of LDN193189, e.g., a salt, hydrate, solvent, ester, or prodrug of LDN193189. In some embodiments, a derivative (e.g., salt) of LDN193189 comprises LDN193189 hydrochloride.

In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises a compound of Formula I from U.S. Patent Publication No. 2011/0053930.

In some embodiments, a medium described herein does not comprise a BMP signaling pathway inhibitor.

TGF-fl Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of TGF-P signaling pathway inhibitors as cell differentiation factors. In some embodiments, the TGF-P signaling pathway comprises TGF-P receptor type I kinase (TGF-P RI) signaling. In some embodiments, the TGF-P signaling pathway inhibitor comprises ALK5 inhibitor II (CAS 446859-33-2, an ATP-competitive inhibitor of TGF-B RI kinase, also known as RepSox, IUPAC Name: 2-[5-(6-methylpyridin-2-yl)- lH-pyrazol-4-yl]-l,5-naphthyridine. In some embodiments, the TGF-P signaling pathway inhibitor is an analog or derivative of ALK5 inhibitor II.

In some embodiments, the analog or derivative of ALK5 inhibitor II (also named “ALK5i”) is a compound of Formula I as described in U.S. Patent Publication No. 2012/0021519, incorporated by reference herein in its entirety.

In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is a TGF-P receptor inhibitor described in U.S. Patent Publication No. 2010/0267731. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein comprises an ALK5 inhibitor described in U.S. Patent Publication Nos. 2009/0186076 and 2007/0142376. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is A 83-01. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is not A 83-01. In some embodiments, the compositions and methods described herein exclude A 83-01. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is SB 431542. In some embodiments, the TGF-P signaling pathway inhibitor is not SB 431542. In some embodiments, the compositions and methods described herein exclude SB 431542. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is D 4476. In some embodiments, the TGF-P signaling pathway inhibitor is not D 4476. In some embodiments, the compositions and methods described herein exclude D 4476. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is GW 788388. In some embodiments, the TGF-P signaling pathway inhibitor is not GW 788388. In some embodiments, the compositions and methods described herein exclude GW 788388. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is LY 364947. In some embodiments, the TGF-P signaling pathway inhibitor is not LY 364947. In some embodiments, the compositions and methods described herein exclude LY 364947. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is LY 580276. In some embodiments, the TGF-P signaling pathway inhibitor is not LY 580276. In some embodiments, the compositions and methods described herein exclude LY 580276. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is SB 525334. In some embodiments, the TGF-P signaling pathway inhibitor is not SB 525334. In some embodiments, the compositions and methods described herein exclude SB 525334. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is SB 505124. In some embodiments, the TGF-P signaling pathway inhibitor is not SB 505124. In some embodiments, the compositions and methods described herein exclude SB 505124. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is SD 208. In some embodiments, the TGF-P signaling pathway inhibitor is not SD 208. In some embodiments, the compositions and methods described herein exclude SD 208. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is GW 6604. In some embodiments, the TGF-P signaling pathway inhibitor is not GW 6604. In some embodiments, the compositions and methods described herein exclude GW 6604. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is GW 788388. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is not GW 788388. In some embodiments, the compositions and methods described herein exclude GW 788388.

From the collection of compounds described above, the following can be obtained from various sources: LY-364947, SB-525334, SD-208, and SB-505124 available from Sigma, P.O. Box 14508, St. Louis, Mo., 63178-9916; 616452 and 616453 available from Calbiochem (EMD Chemicals, Inc.), 480 S. Democrat Road, Gibbstown, N.J., 08027; GW788388 and GW6604 available from GlaxoSmithKline, 980 Great West Road, Brentford, Middlesex, TW8 9GS, United Kingdom; LY580276 available from Lilly Research, Indianapolis, Ind. 46285; and SM16 available from Biogen Idee, P.O. Box 14627, 5000 Davis Drive, Research Triangle Park, N.C., 27709-4627.

In some embodiments, a medium described herein does not comprise a TGF-P signaling pathway inhibitor. WNT Signaling Pathway

Aspects of the disclosure relate to the use of activators of the WNT signaling pathway as cell differentiation factors.

In some embodiments, the WNT signaling pathway activator in the methods and compositions provided herein comprises CHIR99021. In some embodiments, the WNT signaling pathway activator in the methods and compositions provided herein comprises a derivative of CHIR99021, e.g., a salt of CHIR99021, e.g., trihydrochloride, a hydrochloride salt of CHIR99021. In some embodiments, the WNT signaling pathway activator in the methods and compositions provided herein comprises Wnt3a recombinant protein. In some embodiments, the WNT signaling pathway activator in the methods and compositions provided herein comprises a glycogen synthase kinase 3 (GSK3) inhibitor. Exemplary GSK3 inhibitors include, without limitation, 3F8, A 1070722, AR-A 014418, BIO, BlO-acetoxime, FRATide, lOZ-Hymenial disine, Indirubin-3 'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, and analogs or derivatives of any of these. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a WNT signaling pathway activator.

In some embodiments, a medium described herein does not comprise a Wnt signaling pathway activator.

Aspects of the disclosure relate to the use of inhibitors of the WNT signaling pathway as P cell differentiation factors.

In some embodiments, the WNT signaling inhibitor is a tankyrase inhibitor that inhibits expression or activity of at least one tankyrase (TNKS) protein. In some embodiments, the at least one tankyrase protein is tankyrase 1 or tankyrase 2. In some embodiments, the WNT signaling inhibitor inhibits binding of a substrate to a nicotinamide subsite or an adenosine subsite, or both, of a tankyrase protein. In some embodiments, the tankyrase inhibitor is AZ 6102, JW55, MN64, IWR-l-endo, TC-E5001, WIKI4, TNKS 22, TNKS 49, 2X-121 (E7449), XAV-939 (XAV), G007-LK, NVP- TNKS656, decemotinib, (VX-509), vismodegib (GDC-0449), IM- 12, GSK429286A, INO-1001, Ofloxacin, TG101209, FG-4592, l-BET-762, LY2157299, MK- 0752, Wnt- C59 (C59), MCI 568, Pacritinib (SB 1518), SB415286, Drocinostat, IWR-l-endo, Norfloxacin, SH-4-54, Nexturastat A, SB216763, UNCO 79, dephnetin, GF109203X, RepSox, Sotrastaurin, SB431542, tofacitinib (CP-690550, Tasocitinib), AG-14361, CI994 (tacedinaline), Ro 31-8220 mesylate, resveratrol, NVP-TNKS656, or YO-01027. In some embodiments, said tankyrase inhibitor is AZ 6102, NVP-TNKS656, or IWR-l-endo. In some embodiments, the tankyrase inhibitor is NVP-TNKS656 (NVP). In some embodiments, the tankyrase inhibitor selectively inhibits tankyrase 1 over tankyrase 2. In some embodiments, the tankyrase inhibitor selectively inhibits tankyrase 2 over tankyrase 1.

In some embodiments, a medium described herein does not comprise a Wnt signaling pathway inhibitor.

Fibroblast Growth Factor (FGF) Family

Aspects of the disclosure relate to the use of growth factors from the FGF family as cell differentiation factors.

In some embodiments, the growth factor from the FGF family in the methods and compositions provided herein comprises keratinocyte growth factor (KGF). The polypeptide sequences of KGF are available to the skilled artisan. An example of human KGF amino acid sequence is provided in GenBank Accession No. AAB21431, provided as SEQ ID NO: 19).

In some embodiments, the growth factor from the FGF family comprises a polypeptide comprises an amino acid sequence that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 19, or a functional fragment thereof. In some embodiments, the growth factor from the FGF family comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 19.

Human KGF amino acid sequence (GenBank Accession AAB21431; SEQ ID NO: 19)

MHKWILTWILPTLLYRSCFHIICL VGTISL ACNDMTPEQMATNVNC S SPERHTRS Y DYMEGGDIRVRRLFCRTQWYLRIDKRGKVKGTQEMKNNYNIMEIRTVAVGIVAI KGVESEFYLAMNKEGKLYAKKECNEDCNFKELILENHYNTYASAKWTHNGGEM FVALNQKGIPVRGKKTKKEQKTAHFLPMAIT

In some embodiments, the growth factor from the FGF family in the methods and composition provided herein comprises FGF2. The polypeptide sequences of FGF2 are available to the skilled artisan. In some embodiments, the growth factor from the FGF family comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF2 polypeptide sequence (GenBank Accession NP— 001997).

In some embodiments, the at least one growth factor from the FGF family in the methods and composition provided herein comprises FGF8B. The polypeptide sequences of FGF8B are available to the skilled artisan. In some embodiments, the growth factor from the FGF family comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF8B polypeptide sequence (GenBank Accession AAB40954).

In some embodiments, the at least one growth factor from the FGF family in the methods and composition provided herein comprises FGF10. The polypeptide sequences of FGF 10 are available to the skilled artisan. In some embodiments, the growth factor from the FGF family comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF 10 polypeptide sequence (GenBank Accession CAG46489).

In some embodiments, the at least one growth factor from the FGF family in the methods and composition provided herein comprises FGF21. The polypeptide sequences of FGF21 are available to the skilled artisan. In some embodiments, the growth factor from the FGF family comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF21 polypeptide sequence (GenBank Accession AAQ89444.1).

In some embodiments, a medium described herein does not comprise a FGF family protein.

Sonic Hedgehog (SHH) Signaling Pathway

Aspects of the disclosure relate to the use of SHH signaling pathway inhibitors as cell differentiation factors.

In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises Santl. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises SANT2. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises SANT3. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises SANT4. In some embodiments, the SHH signaling pathway inhibitor comprises Cur61414. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises forskolin. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises tomatidine. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises AY9944. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises triparanol. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises compound A or compound B (as disclosed in U.S. Pub. No. 2004/0060568). In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises a steroidal alkaloid that antagonizes hedgehog signaling (e.g., cyclopamine or a derivative thereof) as disclosed in U.S. Pub. No. 2006/0276391. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a SHH signaling pathway inhibitor.

Rho Kinase (ROCK) Signaling Pathway

Aspects of the disclosure relate to the use of ROCK signaling pathway inhibitors (ROCK inhibitors) as cell differentiation factors.

In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises Y-27632 or Thiazovivin. In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises Thiazovivin. In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises Y-27632. In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises the following compound or a derivative thereof

In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises the following compound or a derivative thereof:

Non-limiting examples of ROCK inhibitor that can be used in the methods and compositions provided herein include Thiazovivin, Y-27632, Fasudil/HA1077, H-1152, Ripasudil, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE-104, Olefins, Isoquinolines, Indazoles, and pyridinealkene derivatives, ROKa inhibitor, XD- 4000, HMN-1152, 4-(l-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012, and quinazoline.

In some embodiments, a medium described herein does not comprise a ROCK inhibitor.

Retinoic Acid Signaling Pathway

Aspects of the disclosure relate to the use of modulators of retinoic acid signaling as cell differentiation factors.

In some embodiments, the modulator of retinoic acid signaling in the methods and composition provided herein comprises an activator of retinoic acid signaling. In some embodiments, the RA signaling pathway activator in the methods and composition provided herein comprises retinoic acid. In some embodiments, the RA signaling pathway activator in the methods and composition provided herein comprises a retinoic acid receptor agonist. Exemplary retinoic acid receptor agonists in the methods and composition provided herein include, without limitation, CD 1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80, BMS 753, tazarotene, adapalene, and CD 2314.

In some embodiments, the modulator of retinoic acid signaling in the methods and composition provided herein comprises an inhibitor of retinoic acid signaling. In some embodiments, the retinoic acid signaling pathway inhibitor comprises DEAB (IUPAC Name: 2-[2-(diethylamino)ethoxy]-3-prop-2-enylbenzaldehyde). In some embodiments, the retinoic acid signaling pathway inhibitor comprises an analog or derivative of DEAB. In some embodiments, the retinoic acid signaling pathway inhibitor in the methods and composition provided herein comprises a retinoic acid receptor antagonist. In some embodiments, the retinoic acid receptor antagonist in the methods and composition provided herein comprises (E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2- naphthalenyl)ethenyl]benzoic acid, (E)-4-[[(5,6-dihydro-5,5-dimethyl-8-phenylethynyl)-2- naphthalenyl] ethenyl ]benzoic acid, (E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(2- naphthalenyl)-2-naphthalenyl]ethenyl]-benzoic acid, and (E)-4-[2-[5,6-dihydro-5,5- dimethyl-8-(4-methoxyphenyl)-2-naphthalenyl]ethenyl]benzoic acid. In some embodiments, the retinoic acid receptor antagonist comprises BMS 195614 (CAS#253310-42-8), ER 50891 (CAS#187400-85-7), BMS 493 (CAS#170355-78-9), CD 2665 (CAS#170355-78-9), LE 135 (CAS#155877-83-l), BMS 453 (CAS #166977-43-1), or MM 11253 (CAS#345952-44-5).

In certain embodiments, the methods, compositions, and kits disclosed herein exclude a modulator of retinoic acid signaling. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a retinoic acid signaling pathway activator. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a retinoic acid signaling pathway inhibitor.

In some embodiments, a medium described herein does not comprise retinoic acid.

Protein Kinase C Activator

Aspects of the disclosure relate to the use of protein kinase C activators as cell differentiation factors. Protein kinase C is one of the largest families of protein kinase enzymes and is composed of a variety of isoforms. Conventional isoforms include a, pi, PII, y; novel isoforms include 5, a, r], 0; and atypical isoforms include and t/ . PKC enzymes are primarily cytosolic but translocate to the membrane when activated. In the cytoplasm, PKC is phosphorylated by other kinases or autophosphorylated. In order to be activated, some PKC isoforms (e.g., PKC-a) require a molecule to bind to the diacylglycerol (“DAG”) binding site or the phosphatidylserine (“PS”) binding site. Others are able to be activated without any secondary binding messengers at all. PKC activators that bind to the DAG site include, but are not limited to, bryostatin, picologues, phorbol esters, aplysiatoxin, and gnidimacrin. PKC activators that bind to the PS site include, but are not limited to, polyunsaturated fatty acids and their derivatives. It is contemplated that any protein kinase C activator that is capable, either alone or in combination with one or more other p cell differentiation factors, of inducing the differentiation of at least one insulin-producing, endocrine cell or precursor thereof into a SC-P cell can be used in the methods, compositions, and kits described herein.

In some embodiments, any of the PKC activators disclosed herein is a PKC activator capable of binding to a DAG binding site on a PKC. In some embodiments, the PKC activator is capable of binding to a Cl domain of a PKC. In some embodiments, the PKC activator is a benzolactam-derivative. In some embodiments, the benzolactamderivative is ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4- pentadienoylamino)benzolactam), which may be referred to herein as TPPB or TPB. In some embodiments, contacting a population of cells with a benzolactam-derivative PKC activator (e.g., TPPB) increases cell yield as compared to a population of cells not treated with the benzolactam-derivative PKC activator. In some embodiments, the PKC activator is a phorbol ester. In some embodiments, the phorbol ester is Phorbol 12, 13 -dibutyrate, which may be referred to herein as PDBU or PdbU. In some embodiments, contacting a population of cells with a benzolactam -derivative PKC activator (e.g., TPPB) increases cell yield as compared to a population of cells treated with a phorbol ester PKC activator (e.g., PdbU). In some embodiments, the PKC activator in the methods and composition provided herein comprises PdbU. In some embodiments, the PKC activator in the methods and composition provided herein comprises TPB. In some embodiments, the PKC activator in the methods and composition provided herein comprises cyclopropanated polyunsaturated fatty acids, cyclopropanated monounsaturated fatty acids, cyclopropanated polyunsaturated fatty alcohols, cyclopropanated monounsaturated fatty alcohols, cyclopropanated polyunsaturated fatty acid esters, cyclopropanated monounsaturated fatty acid esters, cyclopropanated polyunsaturated fatty acid sulfates, cyclopropanated monounsaturated fatty acid sulfates, cyclopropanated polyunsaturated fatty acid phosphates, cyclopropanated monounsaturated fatty acid phosphates, macrocyclic lactones, DAG derivatives, isoprenoids, octylindolactam V, gnidimacrin, iripallidal, ingenol, napthalenesulfonamides, diacylglycerol kinase inhibitors, fibroblast growth factor 18 (FGF-18), insulin growth factor, hormones, and growth factor activators, as described in WIPO Pub. No. WO/2013/071282. In some embodiments, the bryostain comprises bryostatin-1, bryostatin-2, bryostatin-3, bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9, bryostatin-10, bryostatin-11, bryostatin-12, bryostatin-13, bryostatin-14, bryostatin-15, bryostatin-16, bryostatin-17, or bryostatin-18. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a protein kinase C activator.

In some embodiments, a medium described herein does not comprise a protein kinase C activator. y-Secretase Inhibitors

Aspects of the disclosure relate to the use of y-secretase inhibitors as cell differentiation factors.

In some embodiments, the y-secretase inhibitor in the methods and composition provided herein comprises XXI. In some embodiments, the y-secretase inhibitor in the methods and composition provided herein comprises DAPT. Additional exemplary y- secretase inhibitors in the methods and composition provided herein include, without limitation, the y-secretase inhibitors described in U.S. Pat. Nos. 7,049,296, 8,481,499, 8,501,813, and WIPO Pub. No. WO/2013/052700. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a y-secretase inhibitor.

In some embodiments, a medium described herein does not comprise a y-secretase inhibitor.

Thyroid Hormone Signaling Pathway Activators

Aspects of the disclosure relate to the use of thyroid hormone signaling pathway activators as cell differentiation factors.

In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises GC-1. In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises an analog or derivative of T3 or GC-1. Exemplary analogs of T3 in the methods and composition provided herein include, but are not limited to, selective and non- selective thyromimetics, TRP selective agonist-GC-1, GC-24,4-Hydroxy-PCB 106, MB07811, MB07344,3,5-diiodothyropropionic acid (DITP A); the selective TR-P agonist GC-1; 3-Iodothyronamine (T(l)AM) and 3,3',5-triiodothyroacetic acid (Triac) (bioactive metabolites of the hormone thyroxine (T(4)); KB-2115 and KB-141; thyronamines; SKF L-94901; DIBIT; 3'-AC-T2; tetraiodothyroacetic acid (Tetrac) and triiodothyroacetic acid (Triac) (via oxidative deamination and decarboxylation of thyroxine [T4] and triiodothyronine [T3] alanine chain), 3, 3 ',5 '-triiodothyronine (rT3) (via T4 and T3 deiodination), 3,3 '-diiodothyronine (3,3'-T2) and 3,5-diiodothyronine (T2) (via T4, T3, and rT3 deiodination), and 3-iodothyronamine (T1AM) and thyronamine (TOAM) (via T4 and T3 deiodination and amino acid decarboxylation), as well as for TH structural analogs, such as 3,5,3 '-triiodothyropropionic acid (Triprop), 3,5-dibromo-3-pyridazinone-l- thyronine (L-940901), N-[3,5-dimethyl-4-(4'-hydroxy-3'-isopropylphenoxy)-phenyl]- oxamic acid (CGS 23425), 3,5-dimethyl-4-[(4'-hydroxy-3'-isopropylbenzyl)- phenoxy]acetic acid (GC-1), 3,5-dichloro-4-[(4-hydroxy-3- isopropylphenoxy)phenyl]acetic acid (KB-141), and 3,5-diiodothyropropionic acid (DITPA).

In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises a prodrug or prohormone of T3, such as T4 thyroid hormone (e.g., thyroxine or L-3,5,3',5'-tetraiodothyronine).

In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein is an iodothyronine composition described in U.S. Pat. No. 7,163,918.

In some embodiments, a medium described herein does not comprise a thyroid hormone.

Epidermal Growth Factor (EGF) Family

Aspects of the disclosure relate to the use of growth factors from the EGF family as cell differentiation factors.

In some embodiments, the at least one growth factor from the EGF family in the methods and composition provided herein comprises betacellulin. An example of human betacellulin amino acid sequence is provided in GenBank Accession No. : AAB25452.1 (SEQ ID NO: 20). In some embodiments, the growth factor from the EGF family used in the compositions and methods described herein comprises an amino acid sequence that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20, or a functional fragment thereof. In some embodiments, the growth factor from the EGF family used in the compositions and methods described herein comprises the amino acid sequence of SEQ ID NO: 20. Human betacellulin amino acid sequence (GenBank: AAB25452.1; SEQ ID NO: 20) MDRAARCSGASSLPLLLALALGLVILHCVVADGNSTRSPETNGLLCGDPEENCAA TTTQSKRKGHFSRCPKQYKHYCIKGRCRFVVAEQTPSCVCDEGYIGARCERVDLF YLRGDRGQILVICLIAVMVVFIILVIGVCTCCHPLRKRRKRKKKEEEMETLGKDITP INEDIEETN

In some embodiments, at least one growth factor from the EGF family in the methods and composition provided herein comprises EGF. Epidermal growth factor (EGF) is a 53 amino acid cytokine which is proteolytically cleaved from a large integral membrane protein precursor. In some embodiments, the growth factor from the EGF family in the methods and composition provided herein comprises a variant EGF polypeptide, for example an isolated epidermal growth factor polypeptide having at least 90% amino acid identity to the human wild-type EGF polypeptide sequence, as disclosed in U.S. Pat. No. 7,084,246. In some embodiments, the growth factor from the EGF family in the methods and composition provided herein comprises an engineered EGF mutant that binds to and agonizes the EGF receptor, as is disclosed in U.S. Pat. No. 8,247,531. Nonlimiting examples of amino acid sequences of growth factors from the EGF family that may be used in the compositions and methods described are provided below. In some embodiments, the growth factor from the EGF family used in the compositions and methods described herein comprises an amino acid sequence that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 21-32, or a functional fragment thereof. In some embodiments, the growth factor from the EGF family used in the compositions and methods described herein comprises the amino acid sequence of any one of SEQ ID NO: 21-32.

Homo sapiens epidermal growth factor (WT) (Genbank: AAS83395.1; SEQ ID NO: 21) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 22) NSDSECPLSHDGYCLHGGVCMYIKAVDRYACNCVVGYIGERCQYRDLTWWGPR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 23) NSDSECPLSHDGYCLHDGVCMYIKALDKYACNCVVGYTGERCQYRDLRWWGRR Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 24) NSNSECPLSHDGYCLHDGVCRYIEALDRYACNCVVGYIGERCQYGDLRWWGRR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 25) NSDSGCPLSHSGYCLHDGVCMYIKALDRYACNCVVGYAGERCQYRDLRWWAR R

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 26) TRGSECPLSHDGYCLHDGVCMYIGALDRYACNCVVGYTGERCQYRDLRWWARR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 27) NSDFGCPLSYDGYCLHDGVCMYIKALDKYACNCVVGYAGERCQYRDLRWWGR R

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 28) SRGSKCPPSHDGYCLHDGVCMYIEALDRYACNCVVGYAGERCQYRDLRWWARR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 29) SSGSECPSSHDGYCLHDGACMYIEALDRYACNCAVGYAGERCQYRDLRWWGRR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 30) SSNSECPPSHDGYCLHDGVCMYIEALDRYACNCVVGYAGERCQYRDLRWWARR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 31) NSYSECPPSYDGYCLHDGVCRYIEALDSYACNCVVGYAGERCQYRDLRWWGRR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 32) SSGSECPLSHDGYCLNDGVCMYIEALDKYACNCVVGYVGERCQYRDLRWWARR

In some embodiments, the at least one growth factor from the EGF family in the methods and composition provided herein is replaced with an agent that activates a signaling pathway in the EGF family. In some embodiments, the growth factor from the EGF family in the methods and composition provided herein comprises a compound that mimics EGF. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a growth factor from the EGF family.

In some embodiments, a medium described herein does not comprise a EGF family growth factor.

Epigenetic Modifying Compounds

Aspects of the disclosure relate to the use of epigenetic modifying compound as cell differentiation factors. The term “epigenetic modifying compound” can refer to a chemical compound that can make epigenetic changes genes, z.e., change gene expression(s) without changing DNA sequences. Epigenetic changes can help determine whether genes are turned on or off and can influence the production of proteins in certain cells, e.g., beta-cells. Epigenetic modifications, such as DNA methylation and histone modification, can alter DNA accessibility and chromatin structure, thereby regulating patterns of gene expression. These processes can be crucial to normal development and differentiation of distinct cell lineages in the adult organism. They can be modified by exogenous influences, and, as such, can contribute to or be the result of environmental alterations of phenotype or pathophenotype. Importantly, epigenetic modification can have a crucial role in the regulation of pluripotency genes, which become inactivated during differentiation. Nonlimiting exemplary epigenetic modifying compound include a DNA methylation inhibitor, a histone acetyltransferase inhibitor, a histone deacetylase inhibitor, a histone methyltransferase inhibitor, a bromodomain inhibitor, or any combination thereof.

In an embodiment, the histone methyltransferase inhibitor is an inhibitor of enhancer of zeste homolog 2 (EZH2). EZH2 is a histone-lysine N-methyltransferase enzyme. Non-limiting examples of an EZH2 inhibitor that can be used in the methods provided herein include 3-deazaneplanocin A (DZNep), EPZ6438, EPZ005687 (an S- adenosylmethionine (SAM) competitive inhibitor), Ell, GSK126, and UNC1999. DZNep can inhibit the hydrolysis of S-adenosyl-L-homocysteine (SAH), which is a product-based inhibitor of all protein methyltransferases, leading to increased cellular concentrations of SAH which in turn inhibits EZH2. DZNep may not be specific to EZH2 and can also inhibit other DNA methyltransferases. GSK126 is a SAM-competitive EZH2 inhibitor that has 150-fold selectivity over EZH1. UNC1999 is an analogue of GSK126, and it is less selective than its counterpart GSK126.

In an embodiment, the histone methyltransferase inhibitor is DZNep. In an embodiment, the HD AC inhibitor is a class I HD AC inhibitor, a class II HD AC inhibitor, or a combination thereof. In an embodiment, the HD AC inhibitor is KD5170 (mercaptoketone-based HDAC inhibitor), MC1568 (class Ila HDAC inhibitor), TMP195 (class Ila HDAC inhibitor), or any combination thereof. In some embodiments, HDAC inhibitor is vorinostat, romidepsin (Istodax), chidamide, panobinostat (farydak), belinostat (PXD101), panobinostat (LBH589), valproic acid, mocetinostat (MGCD0103), abexinostat (PCI-24781), entinostat (MS-275), SB939, resminostat (4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), HBI-8000, (a benzamide HDI), kevetrin, CUDC- 101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, sulforaphane, or any variant thereof.

In some embodiments, a medium described herein does not comprise an epigenetic modifying compound.

Protein Kinase Inhibitors

Aspects of the disclosure relate to the use of protein kinase inhibitors as cell differentiation factors.

In some embodiments, the protein kinase inhibitor in the methods and composition provided herein comprises staurosporine. In some embodiments, the protein kinase inhibitor in the methods and composition provided herein comprises an analog of staurosporine. Exemplary analogs of staurosporine in the methods and composition provided herein include, without limitation, Ro-31-8220, a bisindolylmal eimide (Bis) compound, 10'-{5 "-[(methoxy carbonyl)amino]-2"-methyl}- phenylaminocarbonylstaurosporine, a staralog (see, e.g., Lopez et al., “Staurosporinederived inhibitors broaden the scope of analog- sensitive kinase technology”, J. Am. Chem. Soc. 2013; 135(48): 18153-18159), and, cgp41251.

In some embodiments, the protein kinase inhibitor in the methods and composition provided herein is an inhibitor of PKCp. In some embodiments, the protein kinase inhibitor in the methods and composition provided herein is an inhibitor of PKCP with the following structure or a derivative, analogue or variant of the compound as follows:

In some embodiments, the inhibitor of PKCP is a GSK-2 compound with the following structure or a derivative, analogue or variant of the compound as follows:

In some embodiments, the inhibitor of PKC in the methods and composition provided herein is a bisindolylmaleimide. Exemplary bisindolylmaleimides include, without limitation, bisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide Hl, hydrochloride, or a derivative, analogue or variant thereof.

In some embodiments, the PKC inhibitor in the methods and composition provided herein is a pseudohypericin, or a derivative, analogue, or variant thereof. In some embodiments, the PKC inhibitor in the methods and composition provided herein is indorublin-3-monoximc, 5-Iodo or a derivative, analogue or variant thereof. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a protein kinase inhibitor.

In some embodiments, a medium described herein does not comprise a protein kinase inhibitor, or more specifically, does not comprise staurosporine.

Acetyl CoA-related metabolite

In some embodiments, a composition (e.g., medium) of the disclosure comprises an acetyl CoA-related metabolite. Metabolism of acetyl -coenzyme A (acetyl-CoA) can confer numerous metabolic functions, including energy production, lipid synthesis, and protein acetylation.

Exemplary acetyl CoA-related metabolites include, but are not limited to acetate, pyruvate, ketogenic amino acids, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine, tryptophan, fatty acids, CoA, Isovaleryl-CoA, and P-hydroxybutyrate. In some embodiments, the acetyl CoA-related metabolite is acetate. In some embodiments, a composition of the disclosure contains two or more different acetyl CoA related metabolites, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different acetyl CoA-related metabolites. In some embodiments, the acetyl CoA-related metabolite is acetate. In some embodiments, a medium described herein does not include an acetyl CoA- related metabolite (e.g., does not include acetate).

Histone deacetylase inhibitor (HDACi)

In some embodiments, a composition (e.g., medium) of the disclosure comprises a histone deacetylase inhibitor (HDACi). Histone deacetylase inhibitors (HDACi) are a class of compounds that increase acetylation of lysine residues on histone proteins as well as other, nonhistone, proteins by inhibiting the activity of HD AC enzymes.

Exemplary histone deacetylase inhibitors (HDACi) include, but are not limited to P- Hydroxybutyrate, butyric acid, class I HDACi, class IIA HDACi, class IIB HDACi, class III HDACi, class IV HDACi, HDAC-1, HD AC-2, HDAC-3, HD AC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HD AC-10, HDAC-11, sirtuins, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, Vorinostat (suberoylanilide hydroxamic acid, SAHA, MK0683), Entinostat (MS-275, SNDX-275), Panobinostat (LBH589, NVP- LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391 A, HDAC-IN-3), BRD3308, BRD3308, Tubastatin A TFA (Tubastatin A trifluoroacetate salt), Tubastatin A, SIS 17, NKL 22, BML-210 (CAY10433), TC-H 106, SR-4370, Belinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR 901228, NSC 630176), MC1568, Givinostat (ITF2357), Dacinostat (LAQ824, NVP-LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), PCI-34051, Droxinostat (NS 41080), Abexinostat (PCI-24781), Abexinostat (PCI-24781, CRA-024781), RGFP966, AR-42 (HDAC-42), Ricolinostat (ACY-1215, Rocilinostat), Valproic acid sodium salt (Sodium valproate), Tacedinaline (CI994, PD-123654, GOE- 5549, Acetyldinaline), Fimepinostat (CUDC-907), Sodium butyrate (NaB), Curcumin, Diferuloylmethane, M344, Tubacin, RG2833 (RGFP109), RG2833 (RGFP109), Resminostat (RAS2410), Divalproex Sodium, Scriptaid (GCK 1026), Sodium Phenylbutyrate, Sinapinic acid (Sinapic acid), TMP269, Santacruzamate A (CAY10683), TMP195 (TFMO 2), Valproic acid (VP A), UFO 10, Tasquinimod (ABR-215050), SKLB- 23bb, Isoguanosine, Iforaphane, BRD73954, Citarinostat (ACY-241, HDAC-IN-2), Suberohydroxamic acid, plitomicin, HPOB, LMK-235, Biphenyl -4-sulfonyl chloride (p- Phenylbenzenesulfonyl, 4-henylbenzenesulfonyl, p-Biphenyl sulfonyl), Nexturastat A, TH34, Tucidinostat (Chidamide, HBI-8000, CS-055), (-)-Parthenolide, WT161, CAY10603, CAY10603, ACY-738, RaddeaninA, Tinostamustine(EDO-SlOl), Domatinostat (4SC-202), and BG45.

In some embodiments, the HDACi is P-Hydroxybutyrate. P-Hydroxybutyric acid is a ketone body that, along with butyric acid, is an agonist of hydroxy carboxylic acid receptor 2 (HCA2), a Gi/o-coupled GPCR. In some embodiments, an HDACi inhibitor is an agonist of hydroxy carboxylic acid receptor 2.

In some embodiments, a medium described herein does not comprise an HDACi (e.g., does not include P-Hydroxybutyrate).

Redox homeostasis regulator

In some embodiments, a composition (e.g., medium) of the disclosure comprises a redoxhomeostasis regulator.

Exemplary redox homeostasis regulators include, but are not limited to taurine, respiratory chain regulators, free radical scavengers, regulators of mitochondrial protein synthesis, allium sulphur compounds, anthocyanins, beta-carotene, catechins, copper, cryptoxanthins, flavonoids, indoles, isoflavonoids, lignans, lutein, lycopene, alpha lipoic acid, ellagic acid, manganese, polyphenols, selenium, glutathione, vitamin A, vitamin C, vitamin E, zinc, superoxide disutases, GSHPx, Prx-I, catalase, and co-enzyme Q10.

In some embodiments, the redox homeostasis regulator is taurine.

In some embodiments, a medium described herein does not comprise a redox homeostasis regulator.

Taurine is a non-proteinogenic B-aminosulfonic acid that can be derived from methionine and cysteine metabolism. In some embodiments, taurine can inhibit ROS generation within the respiratory chain.

In some embodiments, a medium described herein does not comprise a redox homeostasis regulator (e.g., does not include taurine).

One carbon metabolism pathway intermediate

In some embodiments, a composition (e.g., medium) of the disclosure comprises a one carbon metabolism pathway intermediate. One-carbon metabolism mediated by folate cofactors, supports multiple physiological processes including amino acid homeostasis (methionine, glycine and serine), biosynthesis of nucleotides (purines, thymidine), epigenetic maintenance, and redox defense. Exemplary one carbon metabolism pathway intermediates include, but are not limited to formate, tetrahydrofolate (THF), 10-formylTHF; 5,10-meTHF; 5,10-meTHF; and 10-formylTHF.

In some embodiments, a medium described herein does not comprise a one carbon metabolism pathway intermediate (e.g., does not include formate).

Glutamine

In some embodiments, a composition (e.g., medium) of the disclosure comprises glutamine. Glutamine (Gin or Q) is an alpha-amino acid. Glutamine can be an essential amino acid within in vitro cell cultures. Glutamine supports the growth of cells, including cells that have high energy demands and synthesize large amounts of proteins and nucleic acids. It is an alternative energy source for rapidly dividing cells and cells that use glucose inefficiently.

In some embodiments, compositions and methods of the disclosure utilize glutamine in a form with increased bioavailability. Because of its chemical instability and importance for cell growth and function, it is important that delivery of L-glutamine be tailored to each unique cell culture process. Glutamine (e.g., L-glutamine) in a free form can be unstable at physiological pH in liquid media, breaking down to ammonium and pyroglutamate at rates that make it a problem in many cell culture and biomanufacturing applications. Therefore, many cell culture media contain stabilized forms of glutamine, including dipeptide forms, such as alanyl-l-glutamine and glycyl-l-glutamine. However, these more stable forms of L-glutamine can also have limited bioavailability, for example, due to a requirement for processing by enzymes, such as cell surface peptidases. Thus in some embodiments, compositions and methods of the disclosure utilize glutamine in a form with increased bioavailability, such as a free glutamine form, such as a non-dipeptide form, a non-alanine-glutamine dipeptide form (e.g., a non-alanyl-l-glutamine form), a non- glycine-glutamine dipeptide form (e.g., a non-glycyl-l-glutamine form), a form that in which glutamine is not conjugated to another amino acid or stabilizing moiety, a monomeric form, a free form, or a combination thereof. In some embodiments, glutamine is provided as a protein hydrolysate.

In some embodiments, a basal media contains glutamine. In some embodiments, glutamine in a form as disclosed herein is added to a media that already contains glutamine. In some embodiments, glutamine in a form as disclosed herein is added to a basal media that contains no glutamine or only low levels of glutamine to increase the bioavailability of glutamine.

In some embodiments, a medium described herein does not comprise glutamine.

Glutamate

In some embodiments, a composition (e.g., medium) of the disclosure comprises glutamate (e.g., L-glutamate). Glutamate can be converted into, for example, g-amino butyric acid (GABA), ornithine, 2-oxoglutarate, glucose or glutathione. Glutamate and metabolites generated therefrom can contribute to, for example, redox homeostasis, cell signaling, nitrogen assimilation, amine catabolism, amino acid biosynthesis, nucleoside biosynthesis, and cofactor production.

In some embodiments, contacting cells with glutamate can improve production of SC-P cells in vitro, for example, providing higher cell yields and recoveries, increased numbers and relative percentages of SC-P cells, enhanced stability and shelf-life of SC-P cells, SC-islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of the SC-P cells in vitro, improved cell viability, improved cell function, reduced immunogenicity after transplantation, or a combination thereof, e.g., relative to a composition that lacks glutamate, or contains a lower concentration of glutamate.

In some embodiments, a medium described herein does not comprise glutamate. Vitamins

In some embodiments, a composition (e.g., medium) of the disclosure comprises one or more vitamins.

Exemplary vitamins include, but are not limited to biotin, vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine) and vitamin B12 (cyanocobalamin). In some embodiments the vitamin modulates fatty acid synthesis. In some embodiments the vitamin modulates branched-chain amino acid metabolism. In some embodiments the vitamin modulates or participates as a co-factor in the TCA cycle, e.g., as a cofactor for pyruvate carboxylase. In some embodiments, the vitamin is biotin. In some embodiments, a composition of the disclosure contains two or more different vitamins, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different vitamins.

In some embodiments, a medium described herein does not comprise a vitamin. Water-soluble synthetic polymer

Water-soluble polymer described herein can refer to any polymer that has hydrophilic property and is soluble in aqueous solution at room temperature. The water- soluble polymer can be either naturally occurring or synthetic. In some embodiments, a water-soluble polymer is an albumin protein (e.g., human serum albumin or bovine serum albumin). In some embodiments, the water-soluble polymer is a water-soluble synthetic polymer. Water-soluble synthetic polymers described herein can refer to any synthetic polymer that has hydrophilic property and is soluble in aqueous solution at room temperature. Water-soluble synthetic polymers applicable in the subject methods and compositions include, but not limited to, poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(Nisopropylacrylamide), and polyacrylamide. The water-soluble synthetic polymer can refer to a polymer compound or a mixture of polymer compounds that may have an idealized chemical formula but a variety of derivatives and/or precursors of the idealized formula, depending on the applicable manufacturing method. In some embodiments, the water-soluble synthetic polymer is used to replace at least partially serum or serum albumin, e.g., BSA or HSA, that is typically utilized in cell differentiation, e.g., differentiation of pancreatic P cells or precursor cells thereof. In some embodiments, the water-soluble synthetic polymer replaces 100% of serum albumin, e.g., BSA or HSA, that is typically utilized in cell differentiation, e.g., differentiation of pancreatic P cells or precursor cells thereof. In some embodiments, the water-soluble synthetic polymer reduces the amount of serum albumin, e.g., BSA or HSA, by at least 20%, 30%, 40%, 50%, 60%, 80%, 90%, 95%, or 99% of that is typically utilized in cell differentiation, e.g., differentiation of pancreatic P cells or precursor cells thereof. In some embodiments, the disclosure provides for a composition comprising a population of any of the cells disclosed herein (e.g., pluripotent stem cells; endoderm cells; primitive gut cells; PDX1- positive, NKX6.1 -negative pancreatic progenitor cells; PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells; insulin-positive cells; and/or pancreatic beta cells) and water soluble polymers, wherein at least 20%, 30%, 40%, 50%, 60%, 80%, 90%, 95%, or 99% of the water soluble polymers in the composition are water-soluble synthetic polymers (e.g., any of the PVA molecules disclosed herein) and wherein the remainder of the water soluble polymers are human serum albumin polypeptides. In some embodiments, the disclosure provides for a composition comprising a population of any of the cells disclosed herein (e.g., pluripotent stem cells; endoderm cells; primitive gut cells; PDX1- positive, NKX6.1 -negative pancreatic progenitor cells; PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells; insulin-positive cells; and/or pancreatic beta cells) and water soluble polymers, wherein no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 80%, 90%, 95%, or 99% of the water soluble polymers are naturally occurring water-soluble polymers (e.g., HSA or BSA). In some embodiments, more than 90%, 95%, 99%, and up to 100% of the water soluble polymers in the composition are water-soluble synthetic polymers (e.g., PVA).

In some embodiments, the water-soluble synthetic polymer applicable to the subject compositions and methods includes polyvinyl alcohol (PVA). Polyvinyl alcohol described herein can refer to a water-soluble synthetic polymer that has an idealized formula [CH2CH(OH)]n, which can be either partially or completed hydrolyzed. In some embodiments, the polyvinyl alcohol is manufactured by either partial or complete hydrolysis of polyvinyl acetate to remove acetate groups. In some embodiments, the polyvinyl alcohol is at most 85% hydrolyzed, e.g., 80% hydrolyzed. The percentage of hydrolyzation measures the approximate percentage (e.g., average percentage) of acetate residue that is hydrolyzed in the polyvinyl acetate precursor polymer. In some embodiments, the polyvinyl alcohol is at least 85% hydrolyzed, e.g., 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. Without wishing to be bound by a certain theory, the polyvinyl alcohol can assume a function of carrier-molecule in the culture medium, which is typically carried out by serum or serum albumin, e.g., HSA. The percentage of hydrolyzation of polyvinyl alcohol can be determined by the manufacturing method utilized to produce the polyvinyl alcohol, e.g., how polyvinyl acetate precursor polymer is converted into polyvinyl alcohol, e.g., conversion by basecatalyzed transesterification with ethanol. In some embodiments, the water-soluble synthetic polymer preparation, e.g., polyvinyl alcohol, that is used in the subject method or present in the subject composition has purity of at least 90%, such as at least 92%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or nearly 100%. Purity of polyvinyl alcohol measures the percentage of synthetic polymer that has the idealized formula [CH2CH(OH)]n in the preparation, which includes polyvinyl alcohol of any percentage of hydrolyzation. Impurity of polyvinyl alcohol preparation can include other polymer materials that do not have the idealized formula [CH2CH(OH)]n, or other organic inorganic materials.

In some embodiments, a medium described herein does not comprise a water- soluble synthetic polymer.

Stem cell derived pancreatic islet cells, compositions and method of use

In some aspects, a population of in vitro differentiated cells (e.g., stem cell-derived pancreatic islet cells) produced using the compositions and methods described herein are also provided. In some embodiments, the population of in vitro differentiated cells (e.g., stem cell-derived pancreatic islet cells) comprises NKX6.1 -positive, ISL1 -positive cells and NKX6.1 -negative, ISLl-positive cells. In some embodiments, the population comprises more NKX6.1 -positive, ISLl-positive cells than NKX6.1 -negative, ISLl- positive cells. In some embodiments, up to 30% of the cells in the population are NKX6.1 -negative, ISLl-positive cells; at least 50% of the cells in the population are NKX6.1 -positive, ISLl-positive cells and wherein less than 20% of the cells in the population are ISL1 -negative cells. In some aspects, methods of using the population of in vitro differentiated cells (e.g., stem cell-derived pancreatic islet cells) described herein to treat diseases (e.g., diabetes) are provided. In some embodiments, the cells in any of the cell populations disclosed herein have not been previously subjected to a cell-sorting process (e.g., affinity binding purification or FACS).

In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1 -positive, ISLl-positive cells and NKX6.1 -negative, ISLl-positive cells. In some embodiments, the population comprises more NKX6.1 -positive, ISLl- positive cells than NKX6.1 -negative, ISLl-positive cells.

In some embodiments, less than 20% of the cells (e.g., about 19%, about 18%, Ibout 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less) in the population are ISLl-negative cells. In some embodiments, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, 1%-11%, 2%-10%, 2%-12%, 4%-12%, 6%-12%, 8%-12%, 2%-8%, 4%-8%, 3%-6% or 3%-5% of the cells in the population are ISLl-negative cells. In some embodiments, 2%-20%, 2%-16%, 2%-12%, 2%-8%, 2%-4%, 4%-20%, 4%-16%, 4%-12%, 4%-8%, 8%-20%, 8%-16%, 8%-12%, 12%-20%, 12%-16%, 16%-20% ofthe cells in the population are ISL1 -negative cells. In some embodiments, less than 20% of the cells in the population are ISL1 -negative cells.

In some embodiments, up to 30% of the cells (e.g., about 15%, about 20%, about 25%, about 30%, or less) in the population are NKX6.1 -negative, ISLl-positive cells. In some embodiments, up to 15%, up to 20%, up to 25%, up to 30%, up to 15%-30%, up to 15%-25%, up to 15%-20%, up to 20%-30%, up to 20%-25%, up to 25%-30% of the cells in the population are NKX6.1 -negative, ISLl-positive cells. In some embodiments, 15%- 30%, 15%-25%, 15%-20%, 20%-30%, 20%-25%, 25%-30% of the cells in the population are NKX6.1 -negative, ISLl-positive cells. In some embodiments, up to 20%-30% of the cells in the population are NKX6.1 -negative, ISLl-positive cells. In some embodiments, up to 30% of the cells in the population are NKX6.1 -negative, ISLl-positive cells.

In some embodiments, at least 60%, at least 65%, at least 70%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISLl-positive cells. In some embodiments, 50- 90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70- 75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%, or 85-90% of the cells in the population are ISLl-positive cells. In some embodiments, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISLl-positive cells. In some embodiments, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% of the cells in the population are ISLl-positive cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises at least 50% (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%) of NXK6.1 -positive, ISLl-positive cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 40%-50%, 40%-60%, 40%-70%, 40%-80%, 40%-90%, 40%-100%, 50%-60%, 50%-70%, 50%-80%, 50%-90%, 50%-100%, 60%-70%, 60%-80%, 60%-90%, 60%- 100%, 70%-80%, 70%-90%, 70%-100%, 80%-90%, 80%-100%, or 90%-100% of NXK6.1 -positive, ISLl-positive cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 50%-70% ofNXK6.1 -positive, ISL1- positive cells. In some embodiments, a population of in vitro differentiated cells described herein comprises at least 50% (e.g., 50%-65%) of NXK6.1 -positive, ISLl-positive cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises about 50%-70% (50%-55%, 50%-60%, 50%-65%, 50%-70%, 55%-60%, 55%- 65%, 55%-70%, 60%-65%, 60%-70%, 65%-70%) of NXK6.1 -positive, ISLl-positive cells and less than 20% of the cells (e.g., about 19%, about 18%, Ibout 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less) in the population are ISL1 -negative cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises cells that express insulin (e.g., cells that express insulin but not glucagon or somatostatin), cells that express glucagon (e.g., cells that express glucagon but not insulin or somatostatin), and cells that express somatostatin (e.g., cells that express somatostatin but not insulin or glucagon). In some embodiments, the expression of insulin in a cell of the compositions suggests that the cell is a SC-P cell. In some embodiments, the expression of glucagon and not expressing somatostatin in a cell of the composition suggests that the cell is a SC-a cell. In some embodiments, the expression of somatostatin and not expressing glucagon in a cell of the composition suggests that the cell is a SC-5 cell. In some embodiments, cells that express insulin are also glucose responsive insulin producing cells.

In some embodiments, cells that express insulin (i.e., SC-P cells) in a population of in vitro differentiated cells described herein exhibit glucose stimulated insulin secretion (GSIS). In some embodiments, cells that express insulin (i.e., SC-P cells) in a population of in vitro differentiated cells described herein further mature (e.g., further maturing in a subject after transplantation) into cells that exhibit glucose stimulated insulin secretion (GSIS).

In some embodiments, a population of in vitro differentiated cells described herein comprises up to 30% (e.g., up to 15%, up to 20%, up to 25%, up to 30%, or less) of stem cell-derived alpha cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 15%-30%, 15%-25%, 15%-20%, 20%-30%, 20%-25%, 25%-30% of stem cell-derived alpha cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 15%, 20%, 25%, 30% of stem cell- derived alpha cells. In some embodiments, a population of in vitro differentiated cells described herein comprises at least 50% (e.g., at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or more) of stem cell-derived beta cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 35%-70%, 35%-65%, 35%-60%, 35%-55%, 35%-50%, 35%-45%, 35%-40%, 40%- 70%, 40%-65%, 40%-60%, 40%-55%, 40%-50%, 40%-45%, 45%-70%, 45%-65%, 45%- 60%, 45%-55%, 45%-50%,50%-70%, 50%-65%, 50%-60%, 50%-55%, 55%-70%, 55%- 65%, 55%-60%, 60%-70%, 60%-65% or 65%-70% of stem cell-derived beta cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 35%, 40%, 45%, 50%, 55%, 60% or 65%, or 70% of stem cell-derived beta cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises: (a) 50%-70%, 50%-65%, 50%-60%, 55%-70%, 55%-65%, 55%-60%, 60%- 70%, 60%-65%, or 65%-70% of the cells in the population of cells express insulin; (b) 5%-30%, 5%-25%, 5%-20%, 5%-15%, 5%-10%, 10%-30%, 10%-25%, 10%-20%, 10%- 15%, 15%-30%, 15%-25%, 15%-20%, 20%-30%, 20%-25%, or 25%-30%of the cells in the population of cells express glucagon but not somatostatin; and/or (c) 3%-20%, 3%- 15%, 3%-12%, 3%-10%, 3%-8%, 3%-5%, 4%-20%, 4%-15%, 4%-12%, 4%-10%, 4%- 8%, 4%-5%, 5%-20%, 5%-15%, 5%-12%, 5%-10%, 5%-8%, 7%-20%, 7%-15%, 7%- 12%, 7%-10%, 9%-20%, 9%-15%, 9%-12%, 8%-10%, 8%-12%, 8%-15%, 8%-20%, 10%-20%, 10%-12%, 10%-l 5%, 12%-20%, 12%-15% or 15%-20% of the cells in the population of cells express somatostatin but not glucagon.

In some embodiments, a population of in vitro differentiated cells described herein comprises: (a) 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%- 90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-90%, 50%-80%, 50%-70%, 50%- 60%, 60%-90%, 60%-80%, 60%-70%, 70%-90%, 70%-80%, 70%-90%, 70%-80%, or 80%-90% of the cells in the population of cells express insulin; (b) 5%-40%, 5%-35%, 5%-30%, 5%-25%, 5%-20%, 5%-15%, 5%-10%, 10%-40%, 10%-35%, 10%-30%, 10%- 25%, 10%-20%, 10%-15%, 15%-40%, 15%-35%, 15%-30%, 15%-25%, 15%-20%, 20%- 40%, 20%-35%, 20%-30%, 20%-25%, 25%-40%, 25%-35%, 25%-30%, 30%-40%, 30%- 35% or 35%-40% of the cells in the population of cells express glucagon but not somatostatin; and (c) 3%-20%, 3%-l 5%, 3%-12%, 3%-10%, 3%-8%, 3%-5%, 4%-20%, 4%-15%, 4%-12%, 4%-10%, 4%-8%, 4%-5%, 5%-20%, 5%-15%, 5%-12%, 5%-10%, 5%-8%, 7%-20%, 7%-15%, 7%-12%, 7%-10%, 9%-20%, 9%-15%, 9%-12%, 8%-10%, 8%-12%, 8%-15%, 8%-20%, 10%-20%, 10%-12%, 10%-15%, 12%-20%, 12%-15% or 15%-20% of the cells in the population of cells express somatostatin but not glucagon.

In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1 -positive, ISLl-positive cells that express lower levels of MAFA than NKX6.1 -positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1 -positive, ISLl-positive cells that express higher levels of MAFB than NKX6.1 -positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1 -positive, ISLl-positive cells that express higher levels of SIX2, HOPX, IAPP and/or UCN3 than NKX6.1 -positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1 -positive, ISLl-positive cells that do not express MAFA. In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1 -positive, ISLl-positive cells that express MAFB. As defined herein, the healthy control adult subject is a non-diabetic subject with a healthy functioning pancreas.

In some embodiments, a population of in vitro differentiated cells described herein comprises a C-peptide content per 1,000 of the in vitro differentiated cells of at least 300 pM (e.g., at least 300 pM, at least 400 pm, at least 500 pm, 300pm-500pm, 300pm-400pm, or 400pm-500pm). In some embodiments, a population of in vitro differentiated cells described herein comprises a glucagon content per 1,000 of the in vitro differentiated cells of at least 100 pM (e.g., at least 100pm, at least 200pm, at least 300 pM, at least 400 pm, at least 500 pm, at least 600pm, at least 700pm, at least 800pm, 100pm-800pm, 100pm- 700pm, 100pm-600pm, 100pm-500pm, 100pm-400pm, 100pm-300pm, 100pm-200pm, 200pm-800pm, 200pm-700pm, 200pm-600pm, 200pm-500pm, 200pm-400pm, 200pm- 300pm, 300pm-800pm, 300pm-700pm, 300pm-600pm, 300pm-500pm, 300pm-400pm, 400pm-800pm, 400pm-700pm, 400pm-600pm, 400pm-500pm, 500pm-800pm, 500pm- 700pm, 500pm-600pm, 600pm-800pm, 600pm-700pm, or 700pm-800pm).

In some embodiments, the percentage of cells expressing a marker provided herein is measured by flow cytometry.

In some embodiments, cells in a population of in vitro differentiated cells described herein form cells clusters. The terms "cluster" and "aggregate" can be used interchangeably, and refer to a group of cells that have close cell-to-cell contact, and in some embodiments, the cells in a cluster can be adhered to one another. A cell cluster comprises a plurality of cells. In some embodiments, a cell cluster comprises at least 10, at least 50, at least 200, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 20,000, at least 30,000, or at least 50,000 cells. In some embodiments, a cell cluster comprises between 10-10,000 cells, between 50-10,000, between 100-10,000, between 100-10,000, between 1,000-10,000, between 500 and 10,000, between 500 and 5,000, between 500 and 2,500, between 500 and 2,000, between 1,000 and 100,000, between 1,000 and 50,000, between 1,000 and 40,000, between 1,000 and 20,000, between 1,000 and 10,000, between 1,000 and 5,000 and between 1,000 and 3,000 cells. In some embodiments, a cell cluster comprises at least 500 cells. In some embodiments, a cell cluster comprises at least 1,000 cells. In some embodiments, a cell cluster comprises at least 2,000 cells. In some embodiments, a cell cluster comprises at least 5,000 cells. In some embodiments, a cell cluster comprises no more than 100,000, no more than 90,000, no more than 80,000, no more than 70,000, no more than 60,000, no more than 50,000, no more than 40,000, no more than 30,000, no more than 20,000, no more than 10,000, no more than 7,000, no more than 5,000, no more than 3,000, no more than 2,000 cells, or no more than 1,000 cells. In some embodiments, the cells in a cluster have not been previously subjected to a cell-sorting process (e.g., affinity binding purification or FACS).

A cell cluster can be in a size similar to an endogenous pancreatic islet. For example, a cell cluster can have a diameter similar to an endogenous pancreatic islet. A diameter of a cell cluster can refer to the largest linear distance between two points on the surface of the cell cluster. In some embodiments, the diameter of a cell cluster is at most 300 pm, 200 pm, 150 pm, 100 pm, 90 pm, 80 pm, 70 pm, 60 pm, 50 pm, or 40 pm. The diameter of a cell cluster can be from about 75 pm to about 250 pm. The diameter of a cell cluster can be at most 100 pm.

In some embodiments, a cell cluster is between about 100 and about 250 microns in diameter (e.g., about 125, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 200, about 210, about 215, about 220, or about 225, microns in diameter). For example, in some embodiments, the cell cluster is between about 125 and about 225, between about 130 and about 160, between about 170 and about 225, between about 140 and about 200, between about 140 and about 170, between about 160 and about 220, between about 170 and about 215, or between about 170 and about 200, microns in diameter.

In some embodiments, any of the cells disclosed herein comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said cells comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said cells comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, any of the cells disclosed herein (e.g., any of the SC- derived beta cells or cells in any of the clusters disclosed herein) comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said at least one gene sequence is the ABO sequence, such that the disruption results in the cell being blood type O. In some embodiments, said at least one gene sequence encodes an MHC- Class I gene. In some embodiments, said MHC-Class I gene encodes beta-2 microglobulin (B2M), HLA-A, HLA-B, or HLA-C. In some embodiments, said at least one gene sequence encodes CIITA. In some embodiments, the cells comprise a genomic disruption in the genes encoding HLA-A and HLA-B, but do not comprise a genomic disruption in the gene encoding HLA-C. In some embodiments, said cells comprise a genomic disruption in a natural killer cell activating ligand gene. In some embodiments, said natural killer cell activating ligand gene encodes intercellular adhesion molecule 1 (ICAM1), CD58, CD155, carcinoembryonic antigen- related cell adhesion molecule 1 (CEACAM1), cell adhesion molecule 1 (CADM1), MHC-Class I polypeptide-related sequence A (MICA), or MHC-Class I polypeptide-related sequence B (MICB). In some embodiments, the cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLADR, relative to stem cells that are not genetically modified. In some embodiments, the cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to stem cells that are not genetically modified. In particular embodiments, the pancreatic islet cells disclosed herein (e.g., the SC-beta cells) have increased expression of PDL1 as compared to endogenous pancreatic islet cells from a healthy control subject. In particular embodiments, the pancreatic islet cells disclosed herein (e.g., the SC-beta cells) have increased expression of CD47 as compared to endogenous pancreatic islet cells from a healthy control subject. In some embodiments, the genomic disruption is induced by use of a gene editing system, e.g., CRISPR Cas technology.

In some embodiments, any of the cells disclosed herein (e.g., any of the stem cells disclosed herein) comprises a “safety switch.” In some embodiments, the safety switches are nucleic acid constructs encoding a switch protein that inducibly causes cell death or stops cell proliferation. In some embodiments, the safety switch is inserted at a defined, specific target locus (e.g., a safe harbor locus) in the genome of an engineered cell, usually at both alleles of the target locus. In some embodiments, the target locus is a safe harbor locus, such as ActB or CLYBL. In some embodiments, the target locus is a gene targeted for disruption (e.g., B2M or CIITA). In some embodiments, the switch protein is activated by contacting with an effective dose of a clinically acceptable orthologous small molecule. In some embodiments, when activated, the safety switch causes the cell to stop proliferation, in some embodiments by activating apoptosis of the cell. In some embodiments, the switch protein comprises herpes-simplex-thymidine-kinase. In some embodiments the switch protein comprises a human caspase protein, e.g. caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, etc. In certain embodiments the protein is human caspase 9. In some embodiments, the caspase protein is fused to a sequence that provides for chemically induced dimerization (CID), in which dimerization occurs only in the presence of the orthologous activating agent. One or more CID domains may be fused to the caspase protein, e.g. two different CID domains may be fused to the caspase protein. In some embodiments the CID domain is a dimerization domain of FKBP or FRB (FKBP- rapamycin-binding) domain of mTOR, which are activated with rapamycin analogs. In some embodiments, the safety switch is any of the safety switches described in WO2021173449 and Jones et al., 2014, Frontiers in Pharmacology, 5(254): 1-8, each of which is incorporated herein in its entirety.

In some embodiments, the population comprises pluripotent stem cells. Pluripotent stem cells may have an ABO blood group type. Blood group types may be selected from A, B, O or AB. In some embodiments, the pluripotent stem cells are ABO blood group type O. In some embodiments, the pluripotent stem cells may be genetically modified such that the cell is ABO blood group type O. In some embodiments, the population further comprises a medium. In some embodiments, the medium comprises a sugar. In some embodiments, the sugar is sucrose or glucose. In some embodiments, the medium comprises the sugar at a concentration of between about 0.05% and about 1.5%. In some embodiments, the medium is a CMRL medium; or wherein the medium is HypoThermosol® FRS Preservation Media.

Some aspects of the present disclosure provide compositions comprising population of in vitro differentiated cells described herein. In some embodiments, a composition comprising population of in vitro differentiated cells described herein are therapeutic compositions. The therapeutic compositions can further comprise a physiologically compatible solution including, for example, artificial cerebrospinal fluid or phosphate-buffered saline. The therapeutic composition can be used to treat, prevent, or stabilize a disease (e.g., diabetes).

In some embodiments, a therapeutic composition further comprises other active agents, such as anti-inflammatory agents, exogenous small molecule agonists, exogenous small molecule antagonists, anti-apoptotic agents, antioxidants, and/or growth factors known to a person having skill in the art.

In some embodiments, a therapeutic composition further comprises a pharmaceutically acceptable carrier (e.g. a medium or an excipient). The term pharmaceutically acceptable carrier (or medium), which may be used interchangeably with the term biologically compatible carrier or medium, can refer to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered therapeutically, but also are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication. Suitable pharmaceutically acceptable carriers can include water, salt solution (such as Ringer's solution), alcohols, oils, gelatins, and carbohydrates, such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, and polyvinyl pyrolidine. Such preparations can be sterilized, and if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring. Pharmaceutical compositions comprising cellular components or products, but not live cells, can be formulated as liquids. Pharmaceutical compositions comprising living nonnative pancreatic P cells can be formulated as liquids, semisolids (e.g., gels, gel capsules, or liposomes) or solids (e.g., matrices, scaffolds and the like). In some embodiments, a therapeutic composition is formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

In some embodiments, a therapeutic composition is optionally manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

In some embodiments, a therapeutic composition comprises one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris- hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

In some embodiments, a therapeutic composition further comprises one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In some embodiments, a therapeutic composition is suitable for administration by any administration route, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra-articular, intraperitoneal, or intracranial), intranasal, buccal, sublingual, or rectal administration routes. In some embodiments, a therapeutic composition is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intraarticular, intraperitoneal, or intracranial) administration.

In some embodiments, a therapeutic composition further comprises one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury- containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

In some embodiments, a therapeutic composition comprises a population of in vitro differentiated cells described herein in an amount that is effective to treat or prevent e.g, diabetes. In some embodiments, a therapeutic composition further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g, aluminum hydroxide); and preservatives.

In some embodiments, a therapeutic composition comprising cells, cell components or cell products may be delivered to the kidney of a patient in one or more of several methods of delivery known in the art. In some embodiments, the compositions are delivered to the kidney e.g., on the renal capsule and/or underneath the renal capsule). In another embodiment, the compositions may be delivered to various locations within the kidney via periodic intraperitoneal or intrarenal injection. Alternatively, the compositions may be applied in other dosage forms known to those skilled in the art, such as pre-formed or in situ-formed gels or liposomes.

In some embodiments, therapeutic compositions comprising live cells in a semisolid or solid carrier may be formulated for surgical implantation on or beneath the renal capsule. It should be appreciated that liquid compositions also may be administered by surgical procedures. In particular cases, semi-solid or solid pharmaceutical compositions may comprise semi-permeable gels, lattices, cellular scaffolds and the like, which may be non-biodegradable or biodegradable. For example, in certain cases, it may be desirable or appropriate to sequester the exogenous cells from their surroundings, yet enable the cells to secrete and deliver biological molecules (e.g., insulin) to surrounding cells or the blood stream. In these cases, cells may be formulated as autonomous implants comprising living cells by a non-degradable, selectively permeable barrier that physically separates the transplanted cells from host tissue. Such implants are sometimes referred to as “immunoprotective,” as they have the capacity to prevent immune cells and macromolecules from killing the transplanted cells in the absence of pharmacologically induced immunosuppression. Various encapsulation devices, degradable gels and networks can be used for the pharmaceutical compositions of the present disclosure. For example, degradable materials particularly suitable for sustained release formulations include biocompatible polymers, such as poly(lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.

In some embodiments, it may be desirable or appropriate to deliver the cells on or in a biodegradable, preferably bioresorbable or bioabsorbable, scaffold or matrix. These typically three-dimensional biomaterials contain the living cells attached to the scaffold, dispersed within the scaffold, or incorporated in an extracellular matrix entrapped in the scaffold. Once implanted into the target region of the body, these implants become integrated with the host tissue, wherein the transplanted cells gradually become established. Examples of scaffold or matrix (sometimes referred to collectively as “framework”) material that may be used in the present disclosure include nonwoven mats, porous foams, or self-assembling peptides. Nonwoven mats, for example, may be formed using fibers comprising a synthetic absorbable copolymer of glycolic and lactic acids (PGA/PLA), foams, and/or poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer.

In some embodiments, the framework is a felt, which can be composed of a multifilament yam made from a bioabsorbable material, e.g., PGA, PLA, PCL copolymers or blends, or hyaluronic acid. The yarn is made into a felt using standard textile processing techniques consisting of crimping, cutting, carding and needling. In another embodiment, cells are seeded onto foam scaffolds that may be composite structures. In many of the abovementioned cases, the framework may be molded into a useful shape. Furthermore, it will be appreciated that non-native pancreatic P cells may be cultured on pre-formed, non- degradable surgical or implantable devices.

In some embodiments, the matrix, scaffold or device may be treated prior to inoculation of cells in order to enhance cell attachment. For example, prior to inoculation, nylon matrices can be treated with 0.1 molar acetic acid and incubated in polylysine, PBS, and/or collagen to coat the nylon. Polystyrene can be similarly treated using sulfuric acid. The external surfaces of a framework may also be modified to improve the attachment or growth of cells and differentiation of tissue, such as by plasma coating the framework or addition of one or more proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), a cellular matrix, and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, among others.

In some aspects, the present disclosure provided devices comprising a population of in vitro differentiated cells described herein. In some embodiments, the population of in vitro differentiated cells described herein form cell clusters. A device can be configured to house the cells described herein which, in particular embodiments, produce and release insulin when implanted into a subject. In some embodiment, a device can further comprise a semipermeable membrane. The semipermeable membrane can be configured to retain the cell cluster in the device and permit passage of insulin secreted by the cells. In some embodiments of the device, the cells can be encapsulated by the semipermeable membrane. The encapsulation can be performed by any technique available to one skilled in the art. The semipermeable membrane can also be made of any suitable material as one skilled in the art would appreciate and verify. For example, the semipermeable membrane can be made of polysaccharide or polycation. In some embodiments, the semipermeable membrane can be made of poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide- co-glycolide) (PLGA), and other polyhydroxyacids, poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyphosphazene, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates, biodegradable polyurethanes, albumin, collagen, fibrin, polyamino acids, prolamines, alginate, agarose, agarose with gelatin, dextran, polyacrylates, ethylene- vinyl acetate polymers and other acyl -substituted cellulose acetates and derivatives thereof, polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolefins, polyethylene oxide, or any combinations thereof. In some embodiments, the semipermeable membrane comprises alginate. In some embodiments, the cells are encapsulated in a microcapsule that comprises an alginate core surrounded by the semipermeable membrane. In some embodiments, the alginate core is modified, for example, to produce a scaffold comprising an alginate core having covalently conjugated oligopeptides with an RGD sequence (arginine, glycine, aspartic acid). In some embodiments, the alginate core is modified, for example, to produce a covalently reinforced microcapsule having a chemoenzymatically engineered alginate of enhanced stability. In some embodiments, the alginate core is modified, for example, to produce membrane-mimetic films assembled by in-situ polymerization of acrylate functionalized phospholipids. In some embodiments, microcapsules are composed of enzymatically modified alginates using epimerases. In some embodiments, microcapsules comprise covalent links between adjacent layers of the microcapsule membrane. In some embodiment, the microcapsule comprises a subsievesize capsule comprising alginate coupled with phenol moieties. In some embodiments, the microcapsule comprises a scaffold comprising alginate-agarose. In some embodiments, the cells are modified with PEG before being encapsulated within alginate. In some embodiments, the cells are encapsulated in photoreactive liposomes and alginate. It should be appreciated that the alginate employed in the microcapsules can be replaced with other suitable biomaterials, including, without limitation, polyethylene glycol (PEG), chitosan, polyester hollow fibers, collagen, hyaluronic acid, dextran with ROD, BHD and polyethylene glycol-diacrylate (PEGDA), poly(MPC-co-n-butyl methacrylate-co-4- vinylphenyl boronic acid) (PMBV) and poly(vinyl alcohol) (PVA), agarose, agarose with gelatin, and multilayer cases of these. In some embodiments, the device provided herein comprise extracorporeal segment, e.g., part of the device can be outside a subject’s body when the device is implanted in the subject. The extracorporeal segment can comprise any functional component of the device, with or without the cells or cell cluster provided herein.

Further provided herein are methods for treating or preventing a disease in a subject. A composition comprising a population of in vitro differentiated cells described herein can be administered into a subject to restore a degree of pancreatic function in the subject. In some embodiments, such composition is transplanted in a subject. The term “transplant” can refer to the placement of cells or cell clusters, any portion of the cells or cell clusters thereof, any compositions comprising cells, cell clusters or any portion thereof, into a subject, by a method or route which results in at least partial localization of the introduced cells or cell clusters at a desired site. In some embodiments, the desired site is the pancreas. In some embodiments, the desired site is a non-pancreatic location, such as in the liver or subcutaneously, for example, in a capsule (e.g., microcapsule) to maintain the implanted cells at the implant location and avoid migration. In some embodiments, the transplanted cells release insulin in an amount sufficient for a reduction of blood glucose levels in the subject.

In some embodiments, a composition comprising a population of in vitro differentiated cells described herein are housed in a device that is implanted in a subject. In some embodiments, a composition comprising a population of in vitro differentiated cells described herein are housed in a device suitable for implantation into a subject. In some embodiments, the device upon implantation in a subject releases insulin while retaining the cells in the device, and facilitates tissue vascularization in and around the device. Exemplary devices are described, for example in WO2018232180, WO20 19068059, WO2019178134, W02020/206150, and W02020/206157, each of which is incorporated-by-reference in its entirety. In some embodiments, a subject is not administered an immune suppression agent during the implantation or vascularization of the device. In some embodiments, the device has a thickness of at least about 300 pm. In some embodiments, the device comprises a membrane comprising a plurality of nodes interconnected by a plurality of fibrils.

In some embodiments, the device comprises a first membrane having a first surface comprising a plurality of channels, and a plurality of second surfaces opposing the first surface; and a second membrane opposite and attached to the plurality of the second surfaces of the first membrane; wherein the first membrane and the second membrane form an enclosed compartment having a surface area to volume ratio of at least about 40 cm-1, and wherein the enclosed compartment provides a volume for housing a cell within the device.

In some embodiments, the enclosed compartment comprises a single continuous open chamber. In some embodiments, the volume is about 8 pL to about 1,000 pL. In some embodiments, the device has at least one of a length and a width of about 0.25 cm to about 3 cm. In some embodiments, the device has a thickness of at least about 300 pm.

In some embodiments, the plurality of channels is generally perpendicular with respect to the first membrane. In some embodiments, the plurality of channels is arranged in a rectilinear array. In some embodiments, the plurality of channels is arranged in a polar array. In some embodiments, the channel has an average diameter of about 400 pm to about 3,000 pm. In some embodiments, the diameter is measured at a narrowest point in the channel. In some embodiments, a center of each channel is separated from the center of another channel by a distance of about 75 pm to about 500 pm. In some embodiments, the channel has a height to diameter ratio of at least about 0.2. In some embodiments, the device has a number of channels per area along a transverse plane, and In some embodiments the number is greater than about 50/cm2.

In some embodiments, at least one of the first membrane and the second membrane comprise a plurality of nodes interconnected by a plurality of fibrils. In some embodiments, at least one of the first membrane and the second membrane comprise PVDF, PTFE, ePTFE, PCL, PE/PES, PP, PS, PMMA, PLGA, PLLA, or any combination thereof. In some embodiments, the device further comprises an opening through the first membrane and/or the second membrane within the channel. In some embodiments, the opening has a concentricity with respect to the channel of at most about 25% the diameter of the channel. In some embodiments is a frame configured to receive the device described herein. In some embodiments, the frame is configured to receive a plurality of cell housing devices. In some embodiments, the frame comprises a flexing mechanism configured to prevent buckling of the cell housing device.

In some embodiments, an implantable encapsulation device comprises an internal volume comprising, disposed therein, a population of in vitro differentiated cells or a composition comprising a population of in vitro differentiated cells described herein described herein. In some embodiments, the implantable encapsulation device comprises at least one membrane that at least partially defines the internal volume. In some embodiments, the at least one membrane includes a first membrane and a second membrane, wherein the first membrane and the second membrane are bonded together to form a seal extending at least partially around the internal volume disposed between the first membrane and the second membrane. In some embodiments, the at least one membrane comprises at least one selected from PVDF, PTFE, ePTFE, PCL, PE/PES, PP, PS, PMMA, PLGA, and PLLA. In some embodiments, the at least one membrane comprises ePTFE.

In some embodiments, a method described herein comprises transplanting a population of in vitro differentiated cells described herein to a subject using any means in the art. For example the methods can comprise transplanting the cell cluster via the intraperitoneal space, portal vein, renal subcapsule, renal capsule, omentum, subcutaneous space, or via pancreatic bed infusion. For example, transplanting can be subcapsular transplanting, intramuscular transplanting, or intraportal transplanting, e.g., intraportal infusion. Immunoprotective encapsulation can be implemented to provide immunoprotection to the cell clusters. In some embodiments, the methods of treatment provided herein can comprise administering one or more immune response modulators for modulating or reducing transplant rejection response or other immune response against the implant (e.g., the cells or the device). Examples of immune response modulator that can be used in the methods can include purine synthesis inhibitors like Azathioprine and Mycophenolic acid, pyrimidine synthesis inhibitors like Leflunomide and Teriflunomide, antifolate like Methotrexate, Tacrolimus, Ciclosporin, Pimecrolimus, Abetimus, Gusperimus, Lenalidomide, Pomalidomide, Thalidomide, PDE4 inhibitor, Apremilast, Anakinra, Sirolimus, Everolimus, Ridaforolimus, Temsirolimus, Umirolimus, Zotarolimus, Anti -thymocyte globulin antibodies, Anti -lymphocyte globulin antibodies, CTLA-4, fragment thereof, and fusion proteins thereof like Abatacept and Belatacept, TNF inhibitor like Etanercept and Pegsunercept, Aflibercept, Alefacept, Rilonacept, antibodies against complement component 5 like Eculizumab, anti-TNF antibodies like Adalimumab, Afelimomab, Certolizumab pegol, Golimumab, Infliximab, and Nerelimomab, antibodies against Interleukin 5 like Mepolizumab, anti-Ig E antibodies like Omalizumab, anti -Interferon antibodies like Faralimomab, anti-IL-6 antibodies like Elsilimomab, antibodies against IL-12 and IL-23 like Lebrikizumab and Ustekinumab, anti-IL-17A antibodies like Secukinumab, anti-CD3 antibodies like Murom onab-CD3, Otelixizumab, Teplizumab, and Visilizumab, anti-CD4 antibodies like Clenoliximab, Keliximab, and Zanolimumab, anti-CDl la antibodies like Efalizumab, anti-CD18 antibodies like Erlizumab, anti-CD20 antibodies like Obinutuzumab, Rituximab, Ocrelizumab and Pascolizumab, anti-CD23 antibodies like Gomiliximab and Lumiliximab, anti-CD40 antibodies like Teneliximab and Toralizumab, antibodies against CD62L/L-selectin like Aselizumab, anti-CD80 antibodies like Galiximab, anti- CD147/Basigin antibodies like Gavilimomab, anti-CD154 antibodies like Ruplizumab, anti-BLyS antibodies like Belimumab and Blisibimod, anti-CTLA-4 antibodies like Ipilimumab and Tremelimumab, anti-CAT antibodies like Bertilimumab, Lerdelimumab, and Metelimumab, anti-Integrin antibodies like Natalizumab, antibodies against Interleukin-6 receptor like Tocilizumab, anti-LFA-1 antibodies like Odulimomab, antibodies against IL-2 receptor/CD25 like Basiliximab, Daclizumab, and Inolimomab, antibodies against T-lymphocyte (Zolimomab aritox) like Atorolimumab, Cedelizumab, Fontolizumab, Maslimomab, Morolimumab, Pexelizumab, Reslizumab, Rovelizumab, Siplizumab, Talizumab, Telimomab aritox, Vapaliximab, and Vepalimomab. As used herein, the term “treating” and “treatment” can refer to administering to a subject an effective amount of a composition (e.g., cell clusters or a portion thereof) so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (e.g., partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. As used herein, the term “treatment” includes prophylaxis.

Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrastemal injection and infusion. In preferred embodiments, the compositions are administered by intravenous infusion or injection.

By “treatment,” “prevention” or “amelioration” of a disease or disorder is meant delaying or preventing the onset of such a disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing down or stopping the progression, aggravation or deterioration the progression or severity of a condition associated with such a disease or disorder. In one embodiment, one or more symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% in comparison to a non-treated subject.

Treatment of Diabetes is determined by standard medical methods. A goal of Diabetes treatment is to bring sugar levels down to as close to normal as is safely possible. Commonly set goals are 80-120 milligrams per deciliter (mg/dl) before meals and 100-140 mg/dl at bedtime. A particular physician may set different targets for the patent, depending on other factors, such as how often the patient has low blood sugar reactions. Useful medical tests include tests on the patient's blood and urine to determine blood sugar level, tests for glycosylated hemoglobin level (HbAlc; a measure of average blood glucose levels over the past 2-3 months, normal range being 4-6%), tests for cholesterol and fat levels, and tests for urine protein level. Such tests are standard tests known to those of skill in the art (see, for example, American Diabetes Association, 1998). A successful treatment program can also be determined by having fewer patients in the program with complications relating to Diabetes, such as diseases of the eye, kidney disease, or nerve disease.

Delaying the onset of diabetes in a subject refers to delay of onset of at least one symptom of diabetes, e.g., hyperglycemia, hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, and hypertension), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, or combinations thereof, for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years or more, and can include the entire lifespan of the subject.

In some embodiments, the reduction of blood glucose levels in the subject, as induced by the transplantation of the cell, or the composition or device provided herein, results in an amount of glucose which is lower than the diabetes threshold. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is human. In some embodiments, the amount of glucose is reduced to lower than the diabetes threshold in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the implanting.

A subject that can be treated by the methods herein can be a human or a nonhuman animal. In some embodiments, a subject can be a mammal. Examples of a subject include but are not limited to primates, e.g., a monkey, a chimpanzee, a bamboo, or a human. In some embodiments, a subject is a human. A subject can be non-primate animals, including, but not limited to, a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a rabbit, and the like. In some embodiments, a subject receiving the treatment is a subject in need thereof, e.g., a human in need thereof.

The terms, “patient” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of Type 1 diabetes, Type 2 Diabetes Mellitus, or pre-diabetic conditions. In addition, the methods described herein can be used to treat domesticated animals and/or pets. A subject can be male or female. A subject can be one who has been previously diagnosed with or identified as suffering from or having Diabetes (e.g., Type 1 or Type 2), one or more complications related to Diabetes, or a pre-diabetic condition, and optionally, but need not have already undergone treatment for the Diabetes, the one or more complications related to Diabetes, or the prediabetic condition. A subject can also be one who is not suffering from Diabetes or a prediabetic condition. A subject can also be one who has been diagnosed with or identified as suffering from Diabetes, one or more complications related to Diabetes, or a pre-diabetic condition, but who show improvements in known Diabetes risk factors as a result of receiving one or more treatments for Diabetes, one or more complications related to Diabetes, or the pre-diabetic condition. Alternatively, a subject can also be one who has not been previously diagnosed as having Diabetes, one or more complications related to Diabetes, or a pre-diabetic condition. For example, a subject can be one who exhibits one or more risk factors for Diabetes, complications related to Diabetes, or a pre-diabetic condition, or a subject who does not exhibit Diabetes risk factors, or a subject who is asymptomatic for Diabetes, one or more Diabetes-related complications, or a pre-diabetic condition. A subject can also be one who is suffering from or at risk of developing Diabetes or a pre-diabetic condition. A subject can also be one who has been diagnosed with or identified as having one or more complications related to Diabetes or a pre- diabetic condition as defined herein, or alternatively, a subject can be one who has not been previously diagnosed with or identified as having one or more complications related to Diabetes or a pre-diabetic condition.

EXAMPLES

The examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1: Consumption of amino acids in different reaction vessels and at different differentiation stages.

This study was designed to assess whether extra metabolites could increase pancreatic islet cell yield and P-cell composition. Specifically, this experiment addresses whether amino acids are consumed uniformly in different reaction vessels for differentiation (e.g., spinners and bioreactors). There are two types of amino acids, essential and non-essential. Non-essential amino acids may be synthesized by the human body, whereas essential amino acids are consumed. Non-essential amino acids include alanine, asparagine, aspartate, glutamate, arginine, glutamine, glycine, proline, tyrosine, serine, and cysteine. Essential amino acids include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.

Cells were seeded and differentiated using a differentiation protocol (Protocol 1, shown in Table 1). After 24-hours had passed since the previous day’s media change, 1 mL of media was collected from a spinner or bioreactor at the following stages: IVP, Stage 0 (StOC) , Stage 1 (StlC), Stage 2 (St2C), Stage 3 (St3C), and Stage 4 (St4C)). The samples, along with 1 mL of unused, fresh media from each of the stages was analyzed for amino acid quantification. The concentration of glycine (FIG. 1 A), histidine (FIG. 3A), serine (FIG. 5 A), and aspartate (FIG. 5B) observed in each sample is plotted for each stage of the differentiation. To determine the amount produced or consumed of an individual amino acid, the concentration of the amino acid in each sample was subtracted from the fresh media value for its respective stage, these values were then divided by the viable cell density for each of the samples to generate glycine produced/Mcell (positive values) or consumed/Mcell (negative values) for each sample. Results of this analysis are shown for glycine (FIG. IB) and histidine (FIG. 3B).

Table 1. Differentiation Media (SI -S5)

To generate fold change values for each sample the amino acid produced/million cells (Mcell) or consumed/Mcell values for each sample were divided by the concentrations in fresh media. An example of fold change values at different stages of differentiation is provided for glycine in FIG. 2A. The dashed line indicates “fresh” media value of “1”, any positive value above this line represents the amount of amino acid (e.g., glycine) produced, whereas any negative value below this line represents the amount of amino acid (e.g., glycine) consumed. The result of this analysis is provided for non- essential amino acids (FIG. 2B) and essential amino acids (FIG. 4).

Some amino acids were both consumed and produced depending on the differentiation stage. For example, glycine was consumed in StOC, and produced in StlC, St2C and St3C (FIG. IB, and FIG. 2A). Compared to other non-essential amino acids, glycine consumption or production was dynamic across different stages of the differentiation. On the other hand, similar to other non-essential amino acids, consumption and production of glycine was similar between reaction vessels (e.g., spinner or bioreactor) (FIG. 2A and FIG. 2B).

Analysis performed on histidine and other essential amino acids demonstrates that consumption of amino acids may be impacted by reaction vessel (e.g., spinner or bioreactor), but equivalent during the differentiation. For example, at the StOC stage histidine was produced in the spinner at greater levels relative to similarly treated samples from the bioreactor (FIG. 3B). The increased essential amino acids consumed in SO correlated with increased cell expansion in the spinners (FIG. 4). For example, in the spinner (more growth condition) at SOC there was 0.93 e6/mL and following a 2.65-fold expansion there was 2.46 e6/mL at the SIC stage. Comparatively, in the bioreactor (less growth condition) at SOC there was 1.01 e6/mL and following a 1.80-fold expansion there were 1.82 e6/mL at the SIC stage. Consumption of essential amino acids in later stages (e.g., StlC, St2C, St3C) were equivalent (FIG. 4).

To determine whether the cells exhaust the supply of any of the amino acids at any stage of the differentiation the metabolite remaining in media (pg/mL) was analyzed at the following stages: IVP, StOC, StlC, St2C and St4C in fresh media, spinners and bioreactor. It was found that in the StlC stage, serine was heavily consumed in both the spinners and bioreactor (FIG. 5 A). On the other hand, glycine switches from being consumed in earlier stages (e.g., IVP and StOC) to being produced during StlC in both the spinner and media. Analysis of the metabolite consumed for serine (FIG. 5C) and glycine (FIG. 5D) at the StlC stage illustrates that consumption or production per cell is similar between the spinner and the bioreactor. It was also found that aspartate is nearly fully consumed in StlC (FIG. 5B).

Example 2: Supplementation of amino acids at different differentiation stages

To determine whether supplementation of amino acids identified in Example 1 altered cellular differentiation outcomes, the following experiments were conducted. At the StlC stage of a differentiation protocol (Protocol 2; see Table 3) aspartate, glycine or serine, either alone or in combination, were added to the culture media. As outlined in Table 2, each of these amino acids were present in the base media (e.g., MCDB) at about 100 pM aspartate, 30 pM glycine and 285 pM serine. The base media (e.g., MCDB) was supplemented with 100 pM aspartate, 270 pM glycine and 300 pM serine to increase their final concentrations by a fold addition of approximately 2x aspartate, lOx glycine, 2x serine. Each amino acid was added to SI base media individually to bring their final concentration to 200 pM, 300 pM and 585 pM for aspartate (see Table 3), glycine and serine respectively. In one condition, the base media was supplemented with a combination of all three (e.g., aspartate, glycine and serine) and the final concentration of all the amino acids were supplemented to achieve the same final concentrations as their respective individual supplementations. A control condition following Protocol 2 only was also performed.

Table 2: Amino acids concentration in base media and supplementation

Table 3. Differentiation Protocol 2 with amino acids supplementation in Stage 1

The amino acid supplemented SI base media were utilized for each SI feed in a Protocol 2 differentiation at 0.3L scale, alongside a control. At SIC, samples of cells were collected, stained with Soxl7 and Oct4 and analyzed by flow cytometry. Control and aspartate supplemented samples are shown in FIG. 6A. In the control, 82.4% of cells were in Soxl7+/Oct4-, while in the aspartate supplemented samples, 75.6% of cells were Soxl7+/Oct4- (FIG. 6A). Table 4 summarizes the results of amino acid supplementation either individually (aspartate, glycine, or serine) or in combination (aspartate, glycine and serine). The results of the analysis of Soxl7+/Oct4- on target cells at SIC show that either individually or in combination, amino acid supplementation had little to no impact on SIC compositions compared to control samples.

Table 4: Percentage of Soxl7+/Oct4- cells at SIC with each amino acid supplementation in SI

At S3C, samples of the cells that were supplemented with different amino acids during SIC were collected and stained with NKX6.1 and PDX1 and analyzed using flow cytometry. Flow results from control and aspartate supplemented samples are illustrated in (FIG. 6B). In the control, 89.3% of cells were in PDX1+/NKX6.1-, while in aspartate supplemented samples 90% of cells were PDX1+/NKX6.1- (FIG. 6B). Table 5 summarizes the results of amino acid supplementation either individually (aspartate, glycine, or serine) or in combination (aspartate, glycine and serine). The results of the analysis of PDX1+/NKX6.1- on target cells at S3C show that either individually or in combination, amino acid supplementation had little to no impact on S3C compositions compared to control samples.

Table 5: Percentage of PDX1+/NKX6.1- cells at S3C with each amino acid supplementation in SI

At S4C, samples of the cells that were supplemented with different amino acids during the SIC step were collected and stained with NKX6.1 and PDX1 and analyzed using flow cytometry. Flow results from control and aspartate supplemented samples are illustrated in (FIG. 6C). In the control, 81.3% of cells were in PDX1+/NKX6.1+, while in the aspartate supplemented samples 77.8% of cells were PDX1+/NKX6.1+ (FIG. 6B).

Table 6 summarizes the results of amino acid supplementation either individually (aspartate, glycine, or serine) or in combination (aspartate, glycine and serine). The results of the analysis of PDX1+/NKX6.1+ on target cells at S4C show that either individually or in combination, amino acid supplementation had little to no impact on S4C compositions compared to control samples.

Table 6: Percentage of PDX1+/NKX6.1+ cells at S4C with each amino acid supplementation in SI

At S5C, samples of the cells that were supplemented with different amino acids during SIC were collected and stained with NKX6.1 and ISL1 and analyzed using flow cytometry. Flow results from control, glycine, and serine supplemented samples are illustrated in (FIG. 6D). Also, a graph of ISL1+/NKX6.1+ (P-like) cells and ISL1+/NKX6.1- (a-like) cells at the S5C stage for each amino acid supplementation either individually (aspartate, glycine, or serine) or in combination (aspartate, glycine and serine) is shown in FIG. 6E. The results of this analysis found that aspartate and glycine supplementation in Stage 1 increased the amount of SC-P-cells, and serine supplementation in Stage 1 promoted a-like cell development. To determine how amino acid supplementation affected yield, cell viability assays were performed. It was found that large aggregates formed in serine and glycine supplemented conditions during S5. There was a decreased yield at harvest of serine and glycine supplemented conditions relative to the control, aspartate supplementation and combination (aspartate, serine and glycine) condition (FIG. 7).

Next, Stage 6 cells were analyzed for cellular composition and yield. The media conditions (DS7) for days 1-4 of Stage 6 are provided in Table 7, and for days 4-11 of stage 6 only 1% HSA was added to DMEM/F12. Three different days during Stage 6 were analyzed (day 4 “D4”, day 7 “D7”, and day 11 “Dl l”). At S6D4, S6D7, and S6D11 samples of cells were collected, stained with NKX6.1 and ISL1 and analyzed by flow cytometry and exemplary results are shown in Figure 15 A. Control cells had a 34.8% population of ISL1+/NKX6.1- (a-like) cells on D4, 36.9% ISL1+/NKX6.1- (a-like) cells on D7 and 39.5% ISL1+/NKX6.1- (a-like) cells on Dl l. Comparatively, cells supplemented with a combination of three amino acids (aspartate, glycine and serine) had a 7.65% population of ISL1+/NKX6.1- (a-like) cells on D4, 9.58% ISL1+/NKX6.1- (a- like) cells on D7 and 11.2% ISL1+/NKX6.1- (a-like) cells on DI 1. Therefore, the cells treated with a combination of three amino acids (aspartate, glycine and serine) had a decrease in a-like cells relative to the Protocol 2 only treated controls. Control cells had a 39.6% population of ISL1+/NKX6.1+ (0-like) cells on D4, 40.2% ISL1+/NKX6.1+ (0- like) cells on D7 and 42.1% ISL1+/NKX6.1+ (0-like) cells on Dl l. Comparatively, cells supplemented with a combination of three amino acids (aspartate, glycine and serine) had a 39.8% population of ISL1+/NKX6.1+ (0-like) cells on D4, 42.9% ISL1+/NKX6.1+ (0- like) cells on D7 and 49.6% ISL1+/NKX6.1+ (0-like) cells on DI 1. The percentage of ISL1+/NKX6.1+ (0-like) cells and ISL1+/NKX6.1 (a-like) cells was also measured for cells that received individual amino acid supplementation either with aspartate, glycine or serine as previously described (FIG. 15B-15D).

Table 7: DS7 Media Conditions

In the next set of experiments, different combinations of amino acids were tested for their effects on yield and P-cell composition. The combinations were as follows: (1) aspartate and glycine, (2) aspartate and serine, and (3) glycine and serine (see Table 3). The control group only received treatment with Protocol 2. Amino acids were present in MCDB base media at about 100 pM aspartate, 30 pM glycine and 285 pM serine. Each amino acid was added to the StlC base media in the previously described combinations to a final concentration of 200 pM aspartate, 300 pM glycine and 585 pM serine respectively. The amino acid supplemented SI base media were utilized for each SI feed in a Protocol 2 differentiation at 0.3L scale, alongside a control. At S5C, samples of cells were collected, stained with NKX6.1 and ISL1 and analyzed by flow cytometry. Control, aspartate/serine and serine/glycine supplemented samples are shown in FIG. 8. The results show that at the S5C stage the control treated sample had a target cell population (e.g., NKX6.1+/ISL1+) of 28.9%, aspartate/serine treated samples had a target cell population (e.g., NKX6.1+/ISL1+) of 34.5%, and serine/glycine treated samples had a target cell population (e.g., NKX6.1+/ISL1+) of 38.8%. These data show that treatment in SI with combinations of aspartate/serine and serine/glycine increased SC-P-cell composition in Stage 5. To determine how amino acid supplementation affected yield, cell viability assays were performed. It was found that combinations of aspartate/serine and serine/glycine had lower yields relative to the control and aspartate/glycine treated samples (FIG. 9A and FIG. 9B).

At S6D11 (Stage 6) samples of cells were collected, stained with NKX6.1 and ISL1 and analyzed by flow cytometry. The results of control, aspartate/serine and serine/glycine supplemented samples are shown in FIG 9C. In the aspartate/serine supplementation condition there was 59.1% SC-P-cell composition. In the serine/glycine supplementation condition there was 59.2% SC-P-cell composition. Therefore, there was an increase in SC-P-cell composition in aspartate/serine and serine/glycine supplemented conditions relative to the control, which had a 49.6% SC-P-cell composition. To determine how amino acid supplementation affected yield, cell viability assays were performed. To determine the change in P-cell over each day of the differentiation at Stage 6, cells were collected and stained with NKX6.1 and ISL1. Next, the on target ISL1+/NKX6.1+ cell% from the control was subtracted from the ISL1+/NKX6.1+ cell% in amino acid supplemented in SI conditions for cells at each day of the S6 differentiation. The results of SC-P-cell gain for aspartate/serine and serine/glycine conditions are shown in FIG. 12 A. The results of SC-P-cell gain for serine/glycine is shown in FIG. 12B. The results show that in serine/glycine treated conditions there is a gain from about 5% SC-P-cell to about SC-12.5% P-cell composition from day 4 to day 11 of the Stage 6 differentiation.

Example 3: Single-cell RNA sequencing analysis determines that Soxl7+ population is more heterogenous than measured by flow cytometry.

To determine how supplementation of amino acids supplementation during the StlC stage could alter cellular differentiation outcomes in later stages (e.g., St5C and St6C), single-cell RNA sequencing was performed. Briefly, at the StlC stage of Protocol 1 differentiation single-cell RNAseq was performed, and cells were mapped according to FIG. 14A. Each cell was labeled and plotted based on the Soxl7 gene expression levels (FIG. 14A). Next, cell identity was determined by the expression of key gene markers (e.g, HHEX, ID4, POU5F1, MSX2, SHISA3, PRTG, KDR and GYPB) (FIG. 14B). The average expression and percent of cells expressing the gene markers was different between cell types (e.g., definitive endoderm, early endoderm, and mesoderm) (FIG. 14C).

Example 4:

Ser/Gly and Asp/Ser supplementation were tested during Stage 1 of the SC-P cell differentiation in the PBS 3MAG bioreactor. Each bioreactor was differentiated using Protocol 2 described in Table 3 using both Nutristem and StemScale as the stem cell expansion media. Where indicated as “Ser/Gly” or “Asp/Ser”, serine, glycine and aspartate was supplemented to the Stage 1 base media to final concentrations of 585 pM, 300 pM and 200 pM, respectively. Upon differentiation to stage 5 and throughout stage 6 (S6D4, D7 and Dl l) flow samples were analyzed for ISL1 and NKX6.1 expression for markers of SC-P cells. The results are shown in FIG. 16A and 16B.

Claims

CLAIMS What is claimed is:

1. An in vitro composition comprising a population of pluripotent stem cells and a medium comprising (i) aspartate at a concentration of at least 120 pM; (ii) glycine at a concentration of at least 40 pM; and/or (iii) serine at a concentration of at least 320 pM.

2. The in vitro composition of claim 1, wherein the medium further comprises a TGF- P ligand.

3. The in vitro composition of claim 1 or claim 2, wherein the TGF-P ligand is activin A.

4. The in vitro composition of claim 2 or claim 3, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 1-50, 1-25, 5-50, 5-25, 5-15, 8-12, 10-1000, 10-500, 10- 250, 10-125, 75-1000, 75-500, 75-250, 75-125, or 90-110 ng/ml.

5. The in vitro composition of claim 4, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 90-110 ng/ml.

6. The in vitro composition of claim 4, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 8-12 ng/ml.

7. The in vitro composition of any one of claims 1-6, wherein the composition further comprises an inhibitor of PI3K/Akt/mTOR signaling.

8. The in vitro composition of claim 7, wherein the inhibitor of PI3K/Akt/mTOR signaling comprises one or more of: GSK-690693, IPI-3063, AZD8055, Omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, Nemiralisib, BYL719, FT113, or Apitolisib, or any analog or derivative thereof.

9. The in vitro composition of claim 8, wherein the inhibitor of PI3K/Akt/mTOR signaling is GSK-690693 or an analog or a derivative thereof.

10. The in vitro composition of claim 9, wherein the GSK-690693, or an analog or a derivative thereof has a concentration of 0.01-1 pM, 0.02-0.8 pM, 0.05-0.5 pM, 0.06-0.2 pM, 0.07-0.15 pM, or 0.08-0.12 pM.

11. The in vitro composition of any one of claims 1-10, wherein the medium further comprises a Wnt signaling pathway activator.

12. The in vitro composition of claim 11, wherein the Wnt signaling pathway activator is a glycogen synthase kinase 3 (GSK3) inhibitor.

13. The in vitro composition of claim 12, wherein the GSK3 inhibitor is CHIR99021.

14. The in vitro composition of any one of claims 11-13, wherein the Wnt signaling pathway activator has a concentration of 0.1-50, 0.1-25, 0.1-10, 0.1-5, 0.5-50, 0.5-25, 0.5- 10, 0.5-5, 1-50, 1-25, 1-10, 1-5, 2-4, or 2-3 pM.

15. The in vitro composition of any one of claims 11-13, wherein the Wnt signaling pathway activator has a concentration of 2-4 pM.

16. The in vitro composition of any one of claims 1-15, wherein the medium further comprises a water-soluble synthetic polymer.

17. The in vitro composition of claim 16, wherein the water-soluble synthetic polymer is polyvinyl alcohol (PVA).

18. The in vitro composition of claim 17, wherein the PVA is at most 85% hydrolyzed, optionally wherein the PVA is about 80% hydrolyzed.

19. The in vitro composition of claim 17 or claim 18, wherein the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the medium.

20. The in vitro composition of any one of claims 1-19, wherein the aspartate has a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160- 300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM, optionally wherein the aspartate has a concentration of 200 pM.

21. The in vitro composition of any one of claims 1-20, wherein the glycine has a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100- 500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM, optionally wherein the glycine has a concentration of 300 pM.

22. The in vitro composition of any one of claims 1-21, wherein the serine has a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500- 600, 320- 1425, 550-650, or 570-620 pM, optionally wherein the serine has a concentration of 585 pM.

23. The in vitro composition of any one of claims 1-19, wherein the aspartate has a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160- 300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and the glycine has a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100- 500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320 or 150-350, optionally wherein the aspartate has a concentration of 200 pM and the glycine has a concentration of 300 pM.

24. The in vitro composition of any one of claims 1-19, wherein the aspartate has a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160- 300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and the serine has a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500- 600, 320- 1425, 550-650, or 570-620 pM, optionally wherein the aspartate has a concentration of 200 pM and serine has a concentration of 585 pM.

25. The in vitro composition of any one of claims 1-19, wherein the glycine has a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100- 500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM and the serine has a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500- 600, 320- 1425, 550-650, or 570-620 pM, optionally wherein the glycine has a concentration of 300 pM and the serine has a concentration of 585 pM.

26. The in vitro composition of any one of claims 1-19, wherein the aspartate has a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160- 300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM, the glycine has a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100- 500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM, and the serine has a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500- 600, 320- 1425, 550-650, or 570-620 pM, optionally wherein the aspartate has a concentration of 200 pM, the glycine has a concentration of 300 pM, and the serine has a concentration of 585 pM.

27. The in vitro composition of any one of claims 1-26, wherein the in vitro composition further comprises definitive endoderm cells.

28. The in vitro composition of any one of claims 1-27, wherein the pluripotent stem cells are embryonic stem cells.

29. The in vitro composition of any one of claims 1-27, wherein the pluripotent stem cells are induced pluripotent stem cells.

30. The in vitro composition of any one of claims 1-29, wherein the pluripotent stem cells are human pluripotent stem cells.

31. The in vitro composition of any one of claims 1-30, wherein the pluripotent stem cells are genetically modified.

32. The in vitro composition of claim 31, wherein the pluripotent stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HL A- A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, relative to cells that are not genetically modified.

33. The in vitro composition of claim 31 or claim 32, wherein the pluripotent stem cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified.

34. The in vitro composition of any one of claims 1-33, wherein the pluripotent stem cells are ABO blood group type O.

35. The in vitro composition of any one of claims 31-33, wherein the pluripotent stem cells have been genetically modified such that the cell is ABO blood group type O.

36. A method comprising culturing a first population of cells in a first medium, wherein: the first population of cells comprises pluripotent stem cells; and the first medium comprises: a): (i) aspartate at a concentration of at least 120 pM;

(ii) glycine at a concentration of at least 40 pM; and/or (iii) serine at a concentration of at least 320 pM, and b) optionally: iv) a Wnt signaling pathway activator, v) a transforming growth factor beta ligand and/or vii) an inhibitor of PI3K/Akt/mTOR signaling.

37. The method of claim 36, wherein the first medium further comprises a transforming growth factor beta (TGF-P) ligand.

38. The method of claim 37, wherein the TGF-P ligand of the first medium is activin A.

39. The method of claim 37 or claim 38, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 1-50, 1-25, 5-50, 5-25, 5-15, 8-12, 10-1000, 10-500, 10-250, 10- 125, 75-1000, 75-500, 75-250, 75-125, or 90-110 ng/ml.

40. The method of claim 39, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 90-110 ng/ml.

41. The method of claim 39, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 8-12 ng/ml.

42. The method of any one of claims 36-41, wherein the first medium further comprises an inhibitor of PI3K/Akt/mT0R signaling.

43. The method of claim 42, wherein the inhibitor of PI3K/Akt/mT0R signaling comprises one or more of: GSK-690693, IPI-3063, AZD8055, Omipalisib, GNE-477, VS- 5584, BYL319, YM201636, PI4KIIIbeta-IN-10, Nemiralisib, BYL719, FT113, or Apitolisib, or any analog or derivative thereof.

44. The method of claim 43, wherein the inhibitor of PI3K/Akt/mT0R signaling is GSK-690693 or an analog or a derivative thereof.

45. The method of claim 44, wherein the GSK-690693, or an analog or a derivative thereof has a concentration of 0.01-1 pM, 0.02-0.8 pM, 0.05-0.5 pM, 0.06-0.2 pM, 0.07- 0.15 pM, or 0.08-0.12 pM.

46. The method of any one of claims 36-45, wherein the first medium further comprises a Wnt signaling pathway activator.

47. The method of claim 46, wherein the Wnt signaling pathway activator is a glycogen synthase kinase 3 (GSK3) inhibitor.

48. The method of claim 47, wherein the GSK3 inhibitor is CHIR99021.

49. The method of any one of claims 46-48, wherein the Wnt signaling pathway activator has a concentration of 0.1-50, 0.1-25, 0.1-10, 0.1-5, 0.5-50, 0.5-25, 0.5-10, 0.5-5, 1-50, 1-25, 1-10, 1-5, 2-4, or 2-3 pM.

50. The method of any one of claims 46-49, wherein the Wnt signaling pathway activator has a concentration of 2-4 pM.

51. The method of any one of claims 36-50, wherein the first medium further comprises a water-soluble synthetic polymer.

52. The method of claim 51, wherein the water-soluble synthetic polymer is polyvinyl alcohol (PVA).

53. The method of claim 52, wherein the PVA is at most 85% hydrolyzed, optionally wherein the PVA is about 80% hydrolyzed.

54. The method of claim 52 or 53, wherein the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the first medium.

55. The method of any one of claims 36-54, wherein the first medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM, optionally wherein the first medium comprises aspartate at a concentration of 200 pM.

56. The method of any one of claims 36-55, wherein the first medium comprises glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300- 600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM, optionally wherein the first medium comprises glycine at a concentration of 300 pM.

57. The method of any one of claims 36-56, wherein the first medium comprises serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320- 600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-400, 320 - 1425, 550-650, or 570-620 pM, optionally wherein the first medium comprises serine at a concentration of 585 pM.

58. The method of any one of claims 36-54, wherein the first medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100- 500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320 or 150-350 pM, optionally wherein the first medium comprises aspartate at a concentration of 200 pM and glycine at a concentration of 300 pM.

59. The method of any one of claims 36-54, wherein the first medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500- 600, 500-400, 320- 1425, 550-650, or 570-620 pM, optionally wherein the first medium comprises aspartate at a concentration of 200 pM and serine at a concentration of 585 pM.

60. The method of any one of claims 36-54, wherein the first medium comprises glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300- 600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320- 600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, , 500-400, 320- 1425, 550-650, or 570-620 pM, optionally wherein the first medium comprises glycine at a concentration of 300 pM and serine at a concentration of 585 pM.

61. The method of any one of claims 36-54, wherein the first medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM, glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100- 500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500- 600, 500-400, 320- 1425, 550-650, or 570-620 pM, optionally wherein the first medium comprises aspartate at a concentration of 200 pM, glycine at a concentration of 300 pM and serine at a concentration of 585 pM.

62. The method of any one of claims 36-61, wherein the first population of cells are cultured in the first medium for a period of 18-48 hours, resulting in a second population of cells, optionally wherein the first population of cells are cultured in the first medium for a period of 24 hours, resulting in a second population of cells.

63. The method of claim 62, further comprising culturing the second population of cells with a second medium comprising: (i) aspartate at a concentration of at least 120 pM; (ii) glycine at a concentration of at least 40 pM; and/or serine at a concentration of at least 320 pM, wherein the second medium does not comprise a Wnt signaling pathway activator.

64. The method of claim 63, wherein the second medium further comprises a TGF-P ligand.

65. The method of claim 64, wherein the TGF-P ligand of the second medium is activin A.

66. The method of claim 65, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 1-50, 1-25, 5-50, 5-25, 5-15, 8-12, 10-1000, 10-500, 10-250, 10-125, 75- 1000, 75-500, 75-250, 75-125, or 90-110 ng/ml.

67. The in vitro composition of claim 66, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 90-110 ng/ml.

68. The in vitro composition of claim 66, wherein the TGF-P ligand (e.g., Activin A) has a concentration of 8-12 ng/ml.

69. The in vitro composition of any one of claims 63- 68, wherein the second medium further comprises an inhibitor of PI3K/Akt/mTOR signaling.

70. The in vitro composition of claim 69, wherein the inhibitor of PI3K/Akt/mTOR signaling comprises one or more of: GSK-690693, IPI-3063, AZD8055, Omipalisib, GNE-477, VS-5584, BYL319, YM201636, PI4KIIIbeta-IN-10, Nemiralisib, BYL719, FT113, or Apitolisib, or any analog or derivative thereof.

71. The in vitro composition of claim 70, wherein the inhibitor of PI3K/Akt/mTOR signaling is GSK-690693 or an analog or a derivative thereof.

72. The in vitro composition of claim 71, wherein the inhibitor of PI3K/Akt/mTOR signaling has a concentration of 0.01-1 pM, 0.02-0.8 pM, 0.05-0.5 pM, 0.06-0.2 pM, 0.07- 0.15 pM, or 0.08-0.12 pM.

73. The method of any one of claims 63-72, wherein the second medium further comprises a water-soluble synthetic polymer.

74. The method of claim 73, wherein the water-soluble synthetic polymer is polyvinyl alcohol (PVA).

75. The method of claim 74, wherein the PVA is at most 85% hydrolyzed, optionally wherein the PVA is about 80% hydrolyzed.

76. The method of claim 74 or 75, wherein the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the second medium.

77. The method of any one of claims 63-76, wherein the second medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM, optionally wherein the second medium comprises aspartate at a concentration of 200 pM.

78. The method of any one of claims 63-77, wherein the second medium comprises glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300- 600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM, optionally wherein the second medium comprises glycine at a concentration of 300 pM.

79. The method of any one of claims 63-78, wherein the second medium comprises serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, SOO- SOO, 500-600, 500-400, 320- 1425, 550-650, or 570-620 pM, optionally wherein the second medium comprises serine at a concentration of 585 pM.

80. The method of any one of claims 63-76, wherein the second medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100- 500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM, optionally wherein the second medium comprises aspartate at a concentration of 200 pM and glycine at a concentration of 300 pM.

81. The method of any one of claims 63-76, wherein the second medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500- 600, 500-400, 320- 1425, 550-650, or 570-620 pM, optionally wherein the second medium comprises aspartate at a concentration of 200 pM and serine at a concentration of 585 pM.

82. The method of any one of claims 63-76, wherein the second medium comprises glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100-500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300- 600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320- 600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500-600, 500-400, 320- 1425, 550-650, or 570-620 pM, optionally wherein the second medium comprises glycine at a concentration of 300 pM and serine at a concentration of 585 pM.

83. The method of any one of claims 63-76, wherein the second medium comprises aspartate at a concentration of 120-1000, 120-800, 120- 500, 120-400, 120-300, 120-220, 120-200, 160-300, 160-250, 160-210, 190-300, 190-250, or 190-210 pM, glycine at a concentration of 40- 600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-80, 100-600, 100- 500, 100-400, 100-300, 100-200, 200-600, 200-400, 200-500, 200-300, 300-600, 300-500, 300-400, 400-600, 400-600, 500-600, 280-320, or 150-350 pM and serine at a concentration of 320-5000, 320-4000, 320-3000, 320-2000, 320-1000, 320-800, 320-600, 320-500, 320-400, 500-5000, 500-4000, 500-3000, 500-2000, 500-1000, 500-800, 500- 600, 500-400, 320- 1425, 550-650, or 570-620 pM, optionally wherein the second medium comprises aspartate at a concentration of 200 pM, glycine at a concentration of 300 pM and serine at a concentration of 585 pM.

84. The method of any one of claims 63-83, wherein the second population of cells are cultured in the second medium for a period of 36-72 hours, resulting in a third population of cells, optionally wherein the second population of cells are cultured in the second medium for a period of 48 hours, resulting in a third population of cells.

85. The method of claim 84, wherein the third population of cells comprises definitive endoderm cells.

86. The method of claim 84 or claim 85, further comprising differentiating the third population of cells into pancreatic endocrine cells.

87. The method of claim 86, wherein the pancreatic endocrine cells comprise beta cells, alpha cells, and delta cells.

88. The method of any one of claims 36-87, wherein the pluripotent stem cells are embryonic stem cells.

89. The method of any one of claims 36-87, wherein the pluripotent stem cells are induced pluripotent stem cells.

90. The method of any one of claims 36-89, wherein the pluripotent stem cells are human pluripotent stem cells.

91. The method of any one of claims 36-90, wherein the pluripotent stem cells are genetically modified.

92. The method of claim 91, wherein the pluripotent stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, relative to cells that are not genetically modified.

93. The method of claim 91 or 92, wherein the pluripotent stem cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified.

94. The method of any one of claims 36-93, wherein the pluripotent stem cells are ABO blood group type O.

95. The method of any one of claims 91-93, wherein the pluripotent stem cells have been genetically modified such that the cell is ABO blood group type O.

96. An in vitro composition comprising a population of in vitro differentiated cells comprising NKX6.1 -positive, ISL1 -positive cells; NKX6.1 -negative, ISL1 -positive cells, and ISLl-negative cells, wherein at least 50% of the cells in the population are NKX6.1- positive, ISLl-positive cells, and wherein less than 20% of the cells are ISLl-negative cells.

97. The in vitro composition of claim 96, wherein 50%-70% of the cells in the population of in vitro differentiated cells are NKX6.1 -positive, ISLl-positive cells.

98. The in vitro composition of claim 96, wherein up to 30% of the cells in the population of in vitro differentiated cells are NKX6.1 -negative, ISLl-positive cells.

99. The in vitro composition of claim 96, wherein up to 20%-30% of the cells in the population of in vitro differentiated cells are NKX6.1 -negative, ISLl-positive cells.

100. The in vitro composition of any one of claims 96-99, further comprising a medium.

101. The in vitro composition of claim 100, wherein the medium comprises human serum albumin.

102. The in vitro composition of claim 100 or 101, wherein the medium comprises glutamine.

103. The in vitro compositions of any one of claims 100-102, wherein the medium comprises any one or more of the following: an inorganic compound, an Alk5 inhibitor, a thyroid hormone receptor beta-specific agonist, a BMP type I receptor inhibitor, a RHO/ROCK pathway inhibitor, a protein kinase inhibitor, or a S-adenosylhomocysteine hydrolase inhibitor.

104. The in vitro composition of any one of claims 100-103, wherein the medium comprises any one or more of the following: ZnSCU, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, or DZNEP.

105. The in vitro composition of any one of claims 100-104, wherein the medium comprises any one or more of L-glutamate, L-camitine, taurine, acetate, beta- hydroxybutarate, biotin or formate.

106. The in vitro composition of any one of claims 100-105, wherein the medium comprises some sugar.

107. The in vitro composition of claim 106, wherein the sugar is sucrose or glucose.

108. The in vitro composition of claim 106 or claim 107, wherein the medium comprises the sugar at a concentration of between about 0.05% and about 1.5%.

109. The in vitro composition of any one of claims 100-108, wherein the medium is a CMRL medium or wherein the medium is HYPOTHERMOSOL® FRS Preservation Media.

110. The in vitro composition of any one of claims 96-109, wherein the population of cells are in a cell cluster.

111. The in vitro composition of 110, wherein the cell cluster is between 125-225 microns, 130-160, 170-225, 140-200, 140-170, 160-220, 170-215, and 170-200 microns in diameter.

112. The in vitro composition of any one of claims 96-111, wherein the population comprises cells that are NKX6.1 -positive, ISL1 -positive, and MAFB-positive cells that do not express MAFA.

113. The composition of any one of claims 96-112, wherein the population of cells is derived from pluripotent stem cells in vitro.

114. The in vitro composition of any one of claims 96-112, wherein the pluripotent stem cells are ABO blood group type O.

115. The in vitro composition of claim 113, wherein the pluripotent stem cells are embryonic stem cells.

116. The in vitro composition of claim 113, wherein the pluripotent stem cells are induced pluripotent stem cells.

117. The in vitro composition of any one of claims 113-116, wherein the pluripotent stem cells are human pluripotent stem cells.

118. The in vitro composition of any one of claims 113-117, wherein the pluripotent stem cells are genetically modified.

119. The in vitro composition of claim 118, wherein the stem cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, relative to cells that are not genetically modified.

120. The in vitro composition of claim 118 or claim 119, wherein the stem cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59 and CTLA, relative to cells that are not genetically modified.

121. The in vitro composition of any one of claims 118-120, wherein the pluripotent stem cells have been genetically modified such that the cell is ABO blood group type O.

122. The in vitro composition of any one of claims 107-121, wherein the pluripotent stem cells are ABO blood group type O.

123. The in vitro composition of any one of claims 96-122, contained in a device for implantation into a subject.

124. An implantable encapsulation device including an internal volume comprising the composition of any one of claims 96-123 disposed therein.

125. The implantable encapsulation device of claim 124, comprising at least one membrane that at least partially defines the internal volume.

126. The implantable encapsulation device of claim 125, wherein the at least one membrane includes a first membrane and a second membrane, wherein the first membrane and the second membrane are bonded together to form a seal extending at least partially around the internal volume disposed between the first membrane and the second membrane.

127. The implantable encapsulation device of claim 125 or claim 126, wherein the at least one membrane comprises at least one selected from PVDF, PTFE, ePTFE, PCL, PE/PES, PP, PS, PMMA, PLGA, and PLLA.

128. The implantable encapsulation device of any one of claims 125-127, wherein the at least one membrane comprises ePTFE.

129. The implantable encapsulation device of any one of claims 125-128, wherein the device has been implanted in a subject having diabetes.

130. The implantable encapsulation device of claim 129, wherein the subject has Type I Diabetes.

131. A method of treating a subject, the method comprising administering to the subject a composition comprising the in vitro composition of any one of claims C1-C28 or implanting the implantable encapsulation device of any one of claims 96-123 in the subject.

132. A method of treating a subject, the method comprising administering to the subject a composition comprising a population in vitro differentiated cells comprising NKX6.1- positive, ISLl-positive cells; NKX6.1 -negative, ISLl-positive cells, and ISLl-negative cells, wherein at least 50% of the cells in the population are NKX6.1 -positive, ISLl- positive cells, and wherein less than 20% of the cells are ISL-negative cells.

133. A method of treating a subject, the method comprising implanting into the subject an implantable encapsulation device comprising a population in vitro differentiated cells comprising NKX6.1 -positive, ISLl-positive cells; NKX6.1 -negative, ISLl-positive cells, and ISLl-negative cells, wherein at least 50% of the cells in the population are NKX6.1- positive, ISLl-positive cells, and wherein less than 20% of the cells are ISL-negative cells.